Serrate fragments and derivatives

ABSTRACT

The present invention relates to sequences of the serrate amino acid sequence as well as fragments thereof, and fragments which retain binding activity are also provided.

This invention was made in part with government support under Grantnumbers GM 29093 and NS 26084 awarded by the Department of Health andHuman Services. The government has certain rights in the invention.

This application is a continuation of application Ser. No. 07/879,038,filed Apr. 30, 1992, now abandoned, which is a continuation-in-part ofapplication Ser. No. 07/791,923 filed Nov. 14, 1991, abandoned, which isa continuation-in-part of application Ser. No. 07/695,189, filed May 3,1991, abandoned, each of which is incorporated by reference herein inits entirety.

1. INTRODUCTION

The present invention relates to the human Notch and Delta genes andtheir encoded products. The invention also relates to sequences (termedherein "adhesive sequences") within the proteins encoded by toporythmicgenes which mediate homotypic or heterotypic binding to sequences withinproteins encoded by toporythmic genes. Such genes include but are notlimited to Notch, Delta, and Serrate.

2. BACKGROUND OF THE INVENTION

Genetic analyses in Drosophila have been extremely useful in dissectingthe complexity of developmental pathways and identifying interactingloci. However, understanding the precise nature of the processes thatunderlie genetic interactions requires a knowledge of the biochemicalproperties of the protein products of the genes in question.

Null mutations in any one of the zygotic neurogenic loci--Notch (N),Delta (Dl), mastermind (mam), Enhancer of Split (E(spl), neuralized(neu), and big brain (bib)--result in hypertrophy of the nervous systemat the expense of ventral and lateral epidermal structures. This effectis due to the misrouting of epidermal precursor cells into a neuronalpathway, and implies that neurogenic gene function is necessary todivert cells within the neurogenic region from a neuronal fate to anepithelial fate. Studies that assessed the effects of laser ablation ofspecific embryonic neuroblasts in grasshoppers (Doe and Goodman 1985,Dev. Biol. 111, 206-219) have shown that cellular interactions betweenneuroblasts and the surrounding accessory cells serve to inhibit theseaccessory cells from adopting a neuroblast fate. Together, these geneticand developmental observations have led to the hypothesis that theprotein products of the neurogenic loci function as components of acellular interaction mechanism necessary for proper epidermaldevelopment (Artavanis-Tsakonas, 1988, Trends Genet. 4, 95-100).

Sequence analyses (Wharton et al., 1985, Cell 43, 567-581; Kidd et al.,1986, Mol. Cell. Biol. 6, 3094-3108; Vassin et al., 1987, EMBO J. 6,3431-3440; Kopczynski et al., 1988, Genes Dev. 2, 1723-1735) have shownthat two of the neurogenic loci, Notch and Delta, appear to encodetransmembrane proteins that span the membrane a single time. The Notchgene encodes a ˜300 kd protein (we use "Notch" to denote this protein)with a large N-terminal extracellular domain that includes 36 epidermalgrowth factor (EGF)--like tandem repeats followed by three othercysteine-rich repeats, designated Notch/lin-12 repeats (Wharton et al.,1985, Cell 43, 567-581; Kidd et al., 1986, Mol. Cell Biol. 6, 3094-3108;Yochem et al., 1988, Nature 335, 547-550). Delta encodes a ˜100 kdprotein (we use "Delta" to denote DLZM, the protein product of thepredominant zygotic and maternal transcripts; Kopczynski et al., 1988,Genes Dev. 2, 1723-1735) that has nine EGF-like repeats within itsextracellular domain (Vassin et al., 1987, EMBO J. 6, 3431-3440;Kopczynski et al., 1988, Genes Dev. 2, 1723-1735). Although little isknown about the functional significance of these repeats, the EGF-likemotif has been found in a variety of proteins, including those involvedin the blood clotting cascade (Furie and Furie, 1988, Cell 53, 505-518).In particular, this motif has been found in extracellular proteins suchas the blood clotting factors IX and X (Rees et al., 1988, EMBO J. 7,2053-2061; Furie and Furie, 1988, Cell 53, 505-518), in other Drosophilagenes (Knust et al., 1987, EMBO J. 761-766; Rothberg et al., 1988, Cell55, 1047-1059), and in some cell-surface receptor proteins, such asthrombomodulin (Suzuki et al., 1987, EMBO J. 6, 1891-1897) and LDLreceptor (Sudhof et al., 1985, Science 228, 815-822). A protein bindingsite has been mapped to the EGF repeat domain in thrombomodulin andurokinase (Kurosawa et al., 1988, J. Biol. Chem 263, 5993-5996; Appellaet al., 1987, J. Biol. Chem. 262, 4437-4440).

An intriguing array of interactions between Notch and Delta mutationshas been described (Vassin, et al., 1985, J. Neurogenet. 2, 291-308;Shepard et al., 1989, Genetics 122, 429-438; Xu et al., 1990, GenesDev., 4, 464-475). A number of genetic studies (summarized in Alton etal., 1989, Dev. Genet. 10, 261-272) has indicated that the gene dosagesof Notch and Delta in relation to one another are crucial for normaldevelopment. A 50% reduction in the dose of Delta in a wild-type Notchbackground causes a broadening of the wing veins creating a "delta" atthe base (Lindsley and Grell, 1968, Publication Number 627, Washington,D.C., Carnegie Institute of Washington). A similar phenotype is causedby a 50% increase in the dose of Notch in a wild-type Delta background(a "Confluens" phenotype; Welshons, 1965, Science 150, 1122-1129). ThisDelta phenotype is partially suppressed by a reduction in the Notchdosage. Recent work in our laboratories has shown that lethalinteractions between alleles that correlate with alterations in theEGF-like repeats in Notch can be rescued by reducing the dose of Delta(Xu et al., 1990, Genes Dev. 4, 464-475). Xu et al. (1990, Genes Dev. 4,464-475) found that null mutations at either Delta or mam suppresslethal interactions between heterozygous combinations of certain Notchalleles, known as the Abruptex (Ax) mutations. Ax alleles are associatedwith missense mutations within the EGF-like repeats of the Notchextracellular domain (Kelley et al., 1987, Cell 51, 539-548; Hartley etal., 1987, EMBO J. 6, 3407-3417).

Notch is expressed on axonal processes during the outgrowth of embryonicneurons (Johansen et al., 1989, J. Cell Biol. 109, 2427-2440; Kidd etal., 1989, Genes Dev. 3, 1113-1129).

A study has shown that certain Ax alleles of Notch can severely alteraxon pathfinding during sensory neural outgrowth in the imaginal discs,although it is not yet known whether aberrant Notch expression in theaxon itself or the epithelium along which it grows is responsible forthis defect (Palka et al., 1990, Development 109, 167-175).

3. SUMMARY OF THE INVENTION

The present invention relates to nucleotide sequences of the human Notchand Delta genes, and amino acid sequences of their encoded proteins, aswell as fragments thereof containing an antigenic determinant or whichare functionally active. The invention is also directed to fragments(termed herein "adhesive fragments"), and the sequences thereof, of theproteins ("toporythmic proteins") encoded by toporythmic genes whichmediate homotypic or heterotypic binding to toporythmic proteins.Toporythmic genes, as used herein, refers to the genes Notch, Delta, andSerrate, as well as other members of the Delta/Serrate family which maybe identified, e.g., by the methods described in Section 5.3, infra.Analogs and derivatives of the adhesive fragments which retain bindingactivity are also provided. Antibodies to human Notch and to adhesivefragments are additionally provided.

In specific embodiments, the adhesive fragment of Notch is that fragmentcomprising the Notch sequence most homologous to Drosophila NotchEGF-like repeats 11 and 12; the adhesive fragment of Delta mediatingheterotypic binding is that fragment comprising the sequence mosthomologous to Drosophila Delta amino acids 1-230; the adhesive fragmentof Delta mediating homotypic binding is that fragment comprising thesequence most homologous to Drosophila Delta amino acids 32-230; and theadhesive fragment of Serrate is that fragment comprising the sequencemost homologous to Drosophila Serrate amino acids 85-283 or 79-282.

3.1. DEFINITIONS

As used herein, the following terms shall have the meanings indicated:

AA=amino acid

EGF=epidermal growth factor

ELR=EGF-like (homologous) repeat

IC=intracellular

PCR=polymerase chain reaction

As used herein, underscoring the name of a gene shall indicate the gene,in contrast to its encoded protein product which is indicated by thename of the gene in the absence of any underscoring. For example,"Notch" shall mean the Notch gene, whereas "Notch" shall indicate theprotein product of the Notch gene.

4. DESCRIPTION OF THE FIGURES

FIG. 1. Expression Constructs and Experimental Design for ExaminingNotch-Delta Interactions. S2 cells at log phase growth were transientlytransfected with one of the three constructs shown. Notch encoded by theMGlla minigene (a cDNA/genomic chimeric construct: cDNA-derivedsequences are represented by stippling, genomically derived sequences bydiagonal-hatching (Ramos et al., 1989, Genetics 123, 337-348)) wasexpressed following insertion into the metallothionein promoter vectorpRmHa-3 (Bunch et al., 1988, Nucl. Acids Res. 16, 1043-1061). Deltaencoded by the Dl1 cDNA (Kopczynski et al., 1988, Genes Dev. 2,1723-1735) was expressed after insertion into the same vector. Theextracellular Notch (ECN1) variant was derived from a genomic cosmidcontaining the complete Notch locus (Ramos et al., 1989, Genetics 123,337-348) by deleting the coding sequence for amino acids 1790-2625 fromthe intracellular domain (denoted by δ; Wharton et al., 1985, Cell 43,567-581), leaving 25 membrane-proximal residues from the wild-typesequence fused to a novel 59 amino acid tail (see ExperimentalProcedures, Section 6.1, infra). This construct was expressed undercontrol of the Notch promoter region. For constructs involving themetallothionein vector, expression was induced with CuSO₄ followingtransfection. Cells were then mixed, incubated under aggregationconditions, and scored for their ability to aggregate using specificantisera and immunofluorescence microscopy to visualize expressingcells. MT, metallothionein promoter; ATG, translation start site; TM,transmembrane domain; 3' N, Notch gene polyadenylation signal; 3' Adh,polyadenylation signal from Adh gene; 5' N, Notch gene promoter region.

FIGS. 2A-2B. Expression of Notch and Delta in Cultured Cells. FIG. 2A:Lysates of nontransfected (S2) and Notch-transfected (N) cells inducedwith 0.7 mM CuSO₄ for 12-16 hr were prepared for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), run on 3%-15% gradientgels, and blotted to nitrocellulose. Notch was visualized using amonoclonal antibody (MAb C17.9C6) against the intracellular domain ofNotch. Multiple bands below the major band at 300 kd may representdegradation products of Notch. FIG. 2B: Lysates of nontransfected (S2)and Delta-transfected (Dl) cells visualized with a monoclonal antibody(MAb 201) against Delta. A single band of .sup.˜ 105 kd is detected. Inboth cases, there is no detectable endogenous Notch or Delta in the S2cell line nor are there cross-reactive species. In each lane, 10 μl ofsample (prepared as described in Experimental Procedures) was loaded.

FIGS. 3A-3I. S2 Cells That Express Notch and Delta Form Aggregates. Inall panels, Notch is shown in green and Delta in red.

FIG. 3A: A single Notch⁺ cell. Note the prominent intracellular stain,including vesicular structures as well as an obviously unstainednucleus.

FIG. 3A: Bright-field micrograph of same field, showing specificity ofantibody staining.

FIG. 3B: A single Delta⁺ cell. Staining is primarily at the cellsurface.

FIG. 3B: Bright-field micrograph of same field.

FIG. 3C: Aggregate of Delta⁺ cells from a 24 hr aggregation experiment.Note against that staining is primarily at the cell surface.

FIGS. 3D-3F: An aggregate of Notch⁺ and Delta⁺ cells formed from a 1:1mixture of singly transfected cell populations that was allowed toaggregate overnight at room temperature. FIG. 3D: shows Notch⁺ cells inthis aggregate; FIG. 3E shows Delta⁺ cells; and FIG. 3F is a doubleexposure showing both cell types. Bands of Notch and Delta are prominentat points of contact between Notch⁺ and Delta⁺ cells (arrows). In FIG.3F, these bands appear yellow because of the coincidence of green andred at these points. The apparently doubly stained single cell (*) isactually two cells (one on top of the other), one expressing Notch andthe other Delta.

FIGS. 3G-3H: Pseudocolor confocal micrographs of Notch⁺ -Delta⁺ cellaggregates. Note that in FIG. 3G extensions (arrows) formed by at leasttwo Delta⁺ cells completely encircle the Notch⁺ cell in the center ofthe aggregate. FIG. 3H shows an aggregate formed from a 2 hr aggregationexperiment performed at 4° C. Intense bands of Notch are apparent withinregions of contact with Delta⁺ cells.

FIG. 3I: An aggregate composed of Delta⁺ cells and cells that expressonly the extracellular domain of Notch (ECN1 construct). Scale bar=10μm.

FIGS. 4A-4F. Notch and Delta are Associated in Cotransfected Cells.Staining for Notch is shown in the left column (FIGS. 4A, 4C, and 4E)and that for Delta is shown in the right column (FIGS. 4B, 4D, and 4F).

FIGS. 4A-4B: S2 cell cotransfected with both Notch and Delta constructs.In general, there was a good correlation between Notch and Deltalocalization at the cell surface (arrows).

FIGS. 4C-4D: Cotransfected cells were exposed to polyclonal anti-Notchantiserum (a 1:250 dilution of each anti-extracellular domain antiserum)for 1 hr at room temperature before fixation and staining with specificantisera. Note punctate staining of Notch and Delta and the correlationof their respective staining (arrows).

FIGS. 4E-4F: Cells cotransfected with the extracellular Notch (ECN1) andDelta constructs, induced, and then patched using anti-Notch polyclonalantisera. There was a close correlation between ECN1 and Delta stainingat the surface as observed for full-length Notch. Scale bar=10 μm.

FIGS. 5A-5D. Coimmunoprecipitation Shows that Delta and Notch areAssociated in Lysates from Transfected S2 and Drosophila EmbryonicCells. In all experiments, Delta was precipitated fromNP-40/deoxycholate lysates using a polyclonal anti-Delta rat antiserumprecipitated with fixed Staph A cells, and proteins in the precipitatedfraction were visualized on Western blots (for details, see ExperimentalProcedures). Lanes 1, 2, 3, and 5: Notch visualized with MAb C17.9C6;Lanes 4 and 6: Delta visualized using MAb 201.

FIG. 5A, lanes 1 and 2 are controls for these experiments. Lane 1 showsa polyclonal anti-Delta immunoprecipitation from cells that expressNotch alone visualized for Notch. No Notch was detectable in thissample, indicating that the polyclonal anti-Delta does not cross-reactwith Notch. Lane 2 shows Notch-Delta cotransfected cellsimmunoprecipitated with Staph A without initial treatment withanti-Delta antiserum and visualized for Notch, demonstrating that Notchis not precipitated nonspecifically by the Staph A or secondaryantibody. Lane 3 shows protein precipitated with anti-Delta antiserumvisualized for Delta (Dl), and lane 4 shows the same sample visualizedfor Notch (N). Lane 4 shows that Notch coprecipitates withimmunoprecipitated Delta. Note that Notch appears as a doublet as istypical for Notch in immunoprecipitates.

FIG. 5B shows the same experiment using embryonic lysates rather thantransfected cell lysates. Lane 5 shows protein precipitated withanti-Delta antiserum visualized for Delta (Dl), and lane 6 shows thesame sample visualized for Notch (N). These lanes demonstrate that Notchand Delta are stably associated in embryo lysates. Bands (in all lanes)below the Delta band are from Staph A (SA) and the anti-Delta antiserumheavy (H) and light (L) chains.

FIGS. 6A-6B. Notch Expression Constructs and the Deletion Mapping of theDelta/Serrate Binding Domain. S2 cells in log phase growth weretransiently transfected with the series of expression constructs shown;the drawings represent the predicted protein products of the variousNotch deletion mutants created. All expression constructs were derivedfrom construct #1 pMtNMg. Transiently transfected cells were mixed withDelta expressing cells from the stably transformed line L49-6-7 or withtransiently transfected Serrate expressing cells, induced with CuSO₄,incubated under aggregation conditions and then scored for their abilityto aggregate using specific antisera and immunofluorescence microscopy.Aggregates were defined as clusters of four or more cells containingboth Notch and Delta/Serrate expressing cells. The values given for %Aggregation refer to the percentage of all Notch expressing cells foundin such clusters either with Delta (Dl) (left column) or with Serrate(Ser) (right column). The various Notch deletion constructs arerepresented diagrammatically with splice lines indicating the ligationjunctions. Each EGF repeat is denoted as a stippled rectangular box andnumbers of the EGF repeats on either side of a ligation junction arenoted. At the ligation junctions, partial EGF repeats produced by thevarious deletions are denoted by open boxes and closed brackets (forexample see #23 ΔCla+EGF(10-12)). Constructs #3-13 represent the ClaIdeletion series. As diagrammed, four of the ClaI sites, in repeats 7, 9,17 and 26, break the repeat in the middle, immediately after the thirdcysteine (denoted by open box repeats; see FIG. 7 for furtherclarification), while the fifth and most 3' site breaks neatly betweenEGF repeats 30 and 31 (denoted by closed box repeat 31; again see FIG.7). In construct #15 split, EGF repeat 14 which carries the split pointmutation, is drawn as a striped box. In construct #33 ΔCla+XEGF(10-13),the Xenopus Notch derived EGF repeats are distinguished from Drosophilarepeats by a different pattern of shading. SP, signal peptide; EGF,epidermal growth factor repeat; N, Notch/lin-12 repeat; TM,transmembrane domain; cdc10, cdc10/ankyrin repeats; PA, putativenucleotide binding consensus sequence; opa, polyglutamine stretch termedopa; Dl, Delta; Ser, Serrate.

FIG. 7. Detailed Structure of Notch Deletion Constructs #19-24: Both EGFRepeats 11 and 12 are Required for Notch-Delta Aggregation. EGF repeats10-13 are diagrammed at the top showing the regular spacing of the sixcysteine residues (C). PCR products generated for these constructs(names and numbers as given in FIGS. 6A-6B) are represented by the heavyblack lines and the exact endpoints are noted relative to the variousEGF repeats. Ability to aggregate with Delta is recorded as (+) or (-)for each construct. The PCR fragments either break the EGF repeats inthe middle, just after the third cysteine in the same place as four outof the five ClaI sites, or exactly in between two repeats in the sameplace as the most C-terminal ClaI site.

FIG. 8. Comparison of Amino Acid Sequence of EGF Repeats 11 and 12 fromDrosophila and Xenopus Notch. The amino acid sequence of EGF repeats 11and 12 of Drosophila Notch (Wharton et al., 1985, Cell 43:567-581; Kiddet al., 1986, Mol. Cell Biol. 6:3094-3108) is aligned with that of thesame two EGF repeats from Xenopus Notch (Coffman et al., 1990, Science249:1438-1441). Identical amino acids are boxed. The six conservedcysteine residues of each EGF repeat and the Ca⁺⁺ binding consensusresidues (Rees et al., 1988, EMBO J. 7:2053-2061) are marked with anasterisk (*). The leucine to proline change found in the Xenopus PCRclone that failed to aggregate is noted underneath.

FIGS. 9A-9C. Constructs Employed in this Study. Schematic diagrams ofthe Delta variants defined in Table IV are shown. Extracellular,amino-proximal terminus is to the left in each case. S, signal peptide;"EGF", EGF-like motifs; M, membrane-spanning helix; H, stop-transfersequence; solid lines, other Delta sequences; hatched lines, neurogliansequences. Arrowheads indicate sites of translatable linker insertions.Sca, ScaI; Nae, NaeI; Bam, BamHI; Bgl, BglII; ELR, EGF-like repeat; Bst,BstEII; Dde, DdeI; Stu, StuI; NG1-NG5, Delta-neuroglian chimeras.

FIG. 9A. Dependence of Aggregation on Input DNA Amounts. Heterotypicaggregation observed using S2 cell populations transiently transfected,respectively, with varied amounts of pMTDl1 DNA (2, 4, 10 or 20μg/plate) that were subsequently incubated under aggregation conditionswith S2 cell populations transiently transfected with a constant amountof pMtNMg DNA (20 μg/plate). Data presented are mean fraction (%) ofDelta cells in aggregates of four or more cells±standard error for eachinput DNA amount (N=3 replicates, except 2 μg and 10 μg inputs for whichN=2). A minimum of 100 Delta-expressing cells were counted for eachreplicate. FIG. 9B, Homotypic aggregation observed using S2 cellpopulations transiently transfected, respectively, with varied amountsof pMTDl1 DNA (2, 4, 10 or 20 μg/plate) that were subsequently incubatedunder aggregation conditions. Data presented are mean fraction (%) ofDelta cells in aggregates of four or more cells±standard error for eachinput DNA amount (N=3 replicates). A minimum of 500 Delta-expressingcells were counted for each replicate.

FIG. 10. Delta-Serrate Amino-Terminal Sequence Alignment. Residues arenumbered on the basis of conceptual translation of Delta (Dl, uppersequence (SEQ ID NO:3); beginning at amino acid 24, ending at amino acid226) and Serrate (Ser, lower sequence (SEQ ID NO:4); beginning at aminoacid 85, ending at amino acid 283) coding sequences. Vertical linesbetween the two sequences indicates residues that are identical withinthe Delta and Serrate sequences, as aligned. Dots represent gaps in thealignment. Boxes enclose cysteine residues within the aligned regions.N1, amino-proximal domain 1; N2, amino-proximal domain 2; N3,amino-proximal domain 3. Translatable insertions associated with STU Breplacement of Delta amino acid 132 (A) with GKIFP! and NAE B insertionof RKIF between Delta amino acid 197 and amino acid 198! constructs,respectively, are depicted above the wild type Delta sequence.

FIGS. 11A-11B. Potential Geometries of Delta-Notch Interactions. FIG.11A, Potential register of Delta (left) and Notch (right) moleculesinteracting between opposing plasma membranes. FIG. 11B, Potentialregister of Delta (left) and Notch (right) molecules interacting withinthe same plasma membranes. ELR, EGF-like repeat; open boxes, EGF-likerepeats; dotted boxes, LNR repeats; solid boxes, membrane-spanninghelices. Delta amino-terminal domain and Delta and Notch intracellulardomains represented by ovals.

FIGS. 12A-12C. Potential Geometries of Delta--Delta Interactions. FIGS.12A-12B, Potential register of Delta molecules interacting betweenopposing plasma membranes. FIG. 12C, Potential register of Deltamolecules interacting within the same plasma membranes. Open boxes,EGF-like repeats; solid boxes, membrane-spanning helices. Deltaamino-terminal extracellular and intracellular domains represented byovals.

FIGS. 13A-13F. Primary Nucleotide Sequence of the Delta cDNA Dl1 (SEQ IDNO:5) and Delta amino acid sequence (SEQ ID NO:6) The DNA sequence ofthe 5'-3' strand of the Dl1 cDNA is shown, which contains a number ofcorrections in comparison to that presented in Kopczynksi et al. (1988,Genes Dev. 2, 1723-1735).

FIG. 14. Primary Nucleotide Sequence of the Neuroglian cDNA 1B7A-250(SEQ ID NO:7). This is the DNA sequence of a portion of the 5'-3' strandof the 1B7A-250 cDNA (A. J. Bieber, pers. comm.; Hortsch et al., 1990,Neuron 4, 697-709). Nucleotide 2890 corresponds to the first nucleotideof an isoleucine codon that encodes amino acid 952 of the conceptuallytranslated neuroglian-long form protein.

FIGS. 15A-15B. Nucleic Acid Sequence Homologies Between Serrate andDelta. A portion of the Drosophila Serrate nucleotide sequence (SEQ IDNO:8), with the encoded Serrate protein sequence (SEQ ID NO:9) writtenbelow, (Fleming et al., 1990, Genes & Dev. 4, 2188-2201 at 2193-94) isshown. The four regions showing high sequence homology with theDrosophila Delta sequence are numbered above the line and indicated bybrackets. The total region of homology spans nucleotide numbers 627through 1290 of the Serrate nucleotide sequence (numbering as in FIG. 4of Fleming et al., 1990, Genes & Dev. 4, 2188-2201).

FIGS. 16A-16C. Primers used for PCR in the Cloning of Human Notch. Thesequence of three primers used for PCR to amplify DNA in a human fetalbrain cDNA library are shown. The three primers, cdc1 (SEQ ID NO:10),cdc2 (SEQ ID NO:11), and cdc3 (SEQ ID NO:12), were designed to amplifyeither a 200 bp or a 400 bp fragment as primer pairs cdc1/cdc2 orcdc1/cdc3, respectively. I: inosine.

FIG. 17. Schematic Diagram of Human Notch Clones. A schematic diagram ofhuman Notch is shown. Heavy bold-face lines below the diagram show thatportion of the Notch sequence contained in each of the four cDNA clones.The location of the primers used in PCR, and their orientation, areindicated by arrows.

FIG. 18. Human Notch Sequences Aligned with Drosophila Notch Sequence.Numbered vertical lines correspond to Drosophila Notch coordinates.Horizontal lines below each map show where clones lie relative tostretches of sequence (thick horizontal lines).

FIGS. 19A-19C. Nucleotide Sequences of Human Notch Contained in PlasmidcDNA Clone hN2k. FIG. 19A: The DNA sequence (SEQ ID NO:13) of a portionof the human Notch insert is shown, starting at the EcoRI site at the 3'end, and proceeding in the 3' to 5' direction. FIG. 19B: The DNAsequence (SEQ ID NO:14) of a portion of the human Notch insert is shown,starting at the EcoRI site at the 5' end, and proceeding in the 5' to 3'direction. FIG. 19C: The DNA sequence (SEQ ID NO:15) of a portion of thehuman Notch insert is shown, starting 3' of the sequence shown in FIG.19B, and proceeding in the 5' to 3' direction. The sequences shown aretentative, subject to confirmation by determination of overlappingsequences.

FIGS. 20A-20D. Nucleotide Sequences of Human Notch Contained in PlasmidcDNA clone hN3k. FIG. 20A: The DNA sequence (SEQ ID NO:16) of a portionof the human Notch insert is shown, starting at the EcoRI site at the 3'end, and proceeding in the 3' to 5' direction. FIG. 20B: The DNAsequence (SEQ ID NO:17) of a portion of the human Notch insert is shown,starting at the EcoRI site at the 5' end, and proceeding in the 5' to 3'direction. FIG. 20C: The DNA sequence (SEQ ID NO:18) of a portion of thehuman Notch insert is shown, starting 3' of the sequence shown in FIG.20B, and proceeding in the 5' to 3' direction. FIG. 20D: The DNAsequence (SEQ ID NO:19) of a portion of the human Notch insert is shown,starting 5' of the sequence shown in FIG. 20A, and proceeding in the 3'to 5' direction. The sequences shown are tentative, subject toconfirmation by determination of overlapping sequences.

FIGS. 21A-21B. Nucleotide Sequences of Human Notch Contained in PlasmidcDNA clone hN4k. FIG. 21A: The DNA sequence (SEQ ID NO:20) of a portionof the human Notch insert is shown, starting at the EcoRI site at the 5'end, and proceeding in the 5' to 3' direction. FIG. 21B: The DNAsequence (SEQ ID NO:21) of a portion of the human Notch insert is shown,starting near the 3' end, and proceeding in the 3' to 5' direction. Thesequences shown are tentative, subject to confirmation by determinationof overlapping sequences.

FIGS. 22A-22D. Nucleotide Sequences of Human Notch Contained in PlasmidcDNA Clone hN5k. FIG. 22A: The DNA sequence (SEQ ID NO:22) of a portionof the human Notch insert is shown, starting at the EcoRI site at the 5'end, and proceeding in the 5' to 3' direction. FIG. 22B: The DNAsequence (SEQ ID NO:23) of a portion of the human Notch insert is shown,starting near the 3' end, and proceeding in the 3' to 5' direction. FIG.22C: The DNA sequence (SEQ ID NO:24) of a portion of the human Notchinsert is shown, starting 3' of the sequence shown in FIG. 22A, andproceeding in the 5' to 3' direction. FIG. 22D: The DNA sequence (SEQ IDNO:25) of a portion of the human Notch insert is shown, starting 5' ofthe sequence shown in FIG. 22B, and proceeding in the 3' to 5'direction. The sequences shown are tentative, subject to confirmation bydetermination of overlapping sequences.

FIGS. 23A-23Q. DNA (SEQ ID NO:31) and Amino Acid (SEQ ID NO:34)Sequences of Human Notch Contained in Plasmid cDNA Clone hN3k.

FIGS. 24A-24G. DNA (SEQ ID NO:33) and Amino Acid (SEQ ID NO:34)Sequences of Human Notch Contained in Plasmid cDNA Clone hN5k.

FIGS. 25A-25C. Comparison of hN5k With Other Notch Homologs. FIG. 25A.Schematic representation of Drosophila Notch. Indicated are the signalsequence (signal), the 36 EGF-like repeats, the three Notch/lin-12repeats, the transmembrane domain (TM), the six CDC10 repeats, the OPArepeat, and the PEST (proline, glutamic acid, serine, threonine)-richregion. FIGS. 25B-25C. Alignment of the deduced amino acid sequence ofhN5k with sequences of other Notch homologs. Amino acids are numbered onthe left side. The cdc10 and PEST-rich regions are both boxed, andindividual cdc10 repeats are marked. Amino acids which are identical inthree or more sequences are highlighted. The primers used to clone hN5kare indicated below the sequences from which they were designed. Thenuclear localization sequence (NLS), casein kinase II (CKII), and cdc2kinase (cdc2) sites of the putative CcN motif of the vertebrate Notchhomologs are boxed. The possible bipartite nuclear targeting sequence(BNTS) and proximal phosphorylation sites of Drosophila Notch are alsoboxed.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleotide sequences of the human Notchand Delta genes, and amino acid sequences of their encoded proteins. Theinvention further relates to fragments (termed herein "adhesivefragments") of the proteins encoded by toporythmic genes which mediatehomotypic or heterotypic binding to toporythmic proteins or adhesivefragments thereof. Toporythmic genes, as used herein, shall mean thegenes Notch, Delta, and Serrate, as well as other members of theDelta/Serrate family which may be identified, e.g. by the methodsdescribed in Section 5.3, infra.

The nucleic acid and amino acid sequences and antibodies thereto of theinvention can be used for the detection and quantitation of mRNA forhuman Notch and Delta and adhesive molecules, to study expressionthereof, to produce human Notch and Delta and adhesive sequences, in thestudy and manipulation of differentiation processes.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention will be divided into the followingsub-sections:

(i) Identification of and the sequences of toporythmic protein domainsthat mediate binding to toporythmic protein domains;

(ii) The cloning and sequencing of human Notch and Delta;

(iii) Identification of additional members of the Delta/Serrate family;

(iv) The expression of toporythmic genes;

(v) Identification and purification of the expressed gene product; and

(vi) Generation of antibodies to toporythmic proteins and adhesivesequences thereof.

5.1. IDENTIFICATION OF AND THE SEQUENCES OF TOPORYTHMIC PROTEIN DOMAINSTHAT MEDIATE BINDING TO TOPORYTHMIC PROTEIN DOMAINS

The invention provides for toporythmic protein fragments, and analogs orderivatives thereof, which mediate homotypic or heterotypic binding (andthus are termed herein "adhesive"), and nucleic acid sequences relatingto the foregoing.

In a specific embodiment, the adhesive fragment of Notch is thatcomprising the portion of Notch most homologous to ELR 11 and 12, i.e.,amino acid numbers 447 through 527 (SEQ ID NO:1) of the Drosophila Notchsequence (see FIG. 8). In another specific embodiment, the adhesivefragment of Delta mediating homotypic binding is that comprising theportion of Delta most homologous to about amino acid numbers 32-230 ofthe Drosophila Delta sequence (SEQ ID NO:6). In yet another specificembodiment, the adhesive fragment of Delta mediating binding to Notch isthat comprising the portion of Delta most homologous to about amino acidnumbers 1-230 of the Drosophila Delta sequence (SEQ ID NO:6). In aspecific embodiment relating to an adhesive fragment of Serrate, suchfragment is that comprising the portion of Serrate most homologous toabout amino acid numbers 85-283 or 79-282 of the Drosophila Serratesequence (see FIG. 10 (SEQ ID NO:4), and FIGS. 15A-15B (SEQ ID NO:9)).

The nucleic acid sequences encoding toporythmic adhesive domains can beisolated from porcine, bovine, feline, avian, equine, or canine, as wellas primate sources and any other species in which homologs of knowntoporythmic genes including but not limited to the following genes (withthe publication of sequences in parentheses): Notch (Wharton et al.,1985, Cell 43, 567-581), Delta (Vassin et al., 1987, EMBO J. 6,3431-3440; Kopczynski et al., 1988, Genes Dev. 2, 1723-1735; notecorrections to the Kopczynski et al. sequence found in FIGS. 13A-13Fhereof (SEQ ID NO:5 and SEQ ID NO:6)) and Serrate (Fleming et al., 1990,Genes & Dev. 4, 2188-2201)! can be identified. Such sequences can bealtered by substitutions, additions or deletions that provide forfunctionally equivalent (adhesive) molecules. Due to the degeneracy ofnucleotide coding sequences, other DNA sequences which encodesubstantially the same amino acid sequence as the adhesive sequences maybe used in the practice of the present invention. These include but arenot limited to nucleotide sequences comprising all or portions of theNotch, Delta, or Serrate genes which are altered by the substitution ofdifferent codons that encode a functionally equivalent amino acidresidue within the sequence, thus producing a silent change. Likewise,the adhesive protein fragments or derivatives thereof, of the inventioninclude, but are not limited to, those containing, as a primary aminoacid sequence, all or part of the amino acid sequence of the adhesivedomains including altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a silent change. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity which acts as a functional equivalent, resulting in asilent alteration. Substitutes for an amino acid within the sequence maybe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

Adhesive fragments of toporythmic proteins and potential derivatives,analogs or peptides related to adhesive toporythmic protein sequences,can be tested for the desired binding activity e.g., by the in vitroaggregation assays described in the examples herein. Adhesivederivatives or adhesive analogs of adhesive fragments of toporythmicproteins include but are not limited to those peptides which aresubstantially homologous to the adhesive fragments, or whose encodingnucleic acid is capable of hybridizing to the nucleic acid sequenceencoding the adhesive fragments, and which peptides and peptide analogshave positive binding activity e.g., as tested in vitro by anaggregation assay such as described in the examples sections infra. Suchderivatives and analogs are envisioned and within the scope of thepresent invention.

The adhesive-protein related derivatives, analogs, and peptides of theinvention can be produced by various methods known in the art. Themanipulations which result in their production can occur at the gene orprotein level. For example, the cloned adhesive protein-encoding genesequence can be modified by any of numerous strategies known in the art(Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). The sequencecan be cleaved at appropriate sites with restriction endonuclease(s),followed by further enzymatic modification if desired, isolated, andligated in vitro. In the production of the gene encoding a derivative,analog, or peptide related to an adhesive domain, care should be takento ensure that the modified gene remains within the same translationalreading frame as the adhesive protein, uninterrupted by translationalstop signals, in the gene region where the desired adhesive activity isencoded.

Additionally, the adhesive-encoding nucleic acid sequence can be mutatedin vitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J.Biol. Chem 253, 6551), use of TAB® linkers (Pharmacia), etc.

Manipulations of the adhesive sequence may also be made at the proteinlevel. Included within the scope of the invention are toporythmicprotein fragments, analogs or derivatives which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄ ; acetylation,formylation, oxidation, reduction; metabolic synthesis in the presenceof tunicamycin; etc.

In addition, analogs and peptides related to adhesive fragments can bechemically synthesized. For example, a peptide corresponding to aportion of a toporythmic protein which mediates the desired aggregationactivity in vitro can be synthesized by use of a peptide synthesizer.

Another specific embodiment of the invention relates to fragments orderivatives of a Delta protein which have the ability to bind to asecond Delta protein or fragment or derivative thereof, but do not bindto Notch. Such binding or lack thereof can be assayed in vitro asdescribed in Section 8. By way of example, but not limitation, such aDelta derivative is that containing an insertion of the tetrapeptideArg-Lys-Ile-Phe (SEQ ID NO:30) between Delta residues 198 and 199 of theDrosophila protein.

5.2. THE CLONING AND SEQUENCING OF HUMAN NOTCH AND DELTA

The invention further relates to the amino acid sequences of human Notchand human Delta and fragments and derivatives thereof which comprise anantigenic determinant (i.e., can be recognized by an antibody) or whichare functionally active, as well as nucleic acid sequences encoding theforegoing. "Functionally active" material as used herein refers to thatmaterial displaying one or more known functional activities associatedwith the full-length (wild-type) protein product, e.g., in the case ofNotch, binding to Delta, binding to Serrate, antigenicity (binding to ananti-Notch antibody), etc.

In specific embodiments, the invention provides fragments of a humanNotch protein consisting of at least 40 amino acids, or of at least 77amino acids. In other embodiments, the proteins of the inventioncomprise or consist essentially of the intracellular domain,transmembrane region, extracellular domain, cdc10 region, Notch/lin-12repeats, or the EGF-homologous repeats, or any combination of theforegoing, of a human Notch protein. Fragments, or proteins comprisingfragments, lacking some or all of the EGF-homologous repeats of humanNotch are also provided.

In other specific embodiments, the invention is further directed to thenucleotide sequences and subsequences of human Notch and human Deltaconsisting of at least 25 nucleotides, at least 50 nucleotides, or atleast 121 nucleotides. Nucleic acids encoding the proteins and proteinfragments described above are also provided, as well as nucleic acidscomplementary to and capable of hybridizing to such nucleic acids. Inone embodiment, such a complementary sequence may be complementary to ahuman Notch cDNA sequence of at least 25 nucleotides, or of at least 121nucleotides. In a preferred aspect, the invention relates to cDNAsequences encoding human Notch or a portion thereof. In a specificembodiment, the invention relates to the nucleotide sequence of thehuman Notch gene or cDNA, in particular, comprising those sequencesdepicted in FIGS. 19A-19C, 20A-20D, 21A-21B and/or 22A-22D (SEQ ID NO:13through NO:25), or contained in plasmids hN3k, hN4k, or hN5k (seeSection 9, infra), and the encoded Notch protein sequences. As isreadily apparent, as used herein, a "nucleic acid encoding a fragment orportion of a Notch protein" shall be construed as referring to a nucleicacid encoding only the recited fragment or portion of the Notch proteinand not other portions of the Notch protein.

In a preferred, but not limiting, aspect of the invention, a human NotchDNA sequence can be cloned and sequenced by the method described inSection 9, infra.

A preferred embodiment for the cloning of human Delta, presented as aparticular example but not by way of limitation follows:

A human expression library is constructed by methods known in the art.For example, human mRNA is isolated, cDNA is made and ligated into anexpression vector (e.g., a bacteriophage derivative) such that it iscapable of being expressed by the host cell into which it is thenintroduced. Various screening assays can then be used to select for theexpressed human Delta product. In one embodiment, selection can becarried out on the basis of positive binding to the adhesive domain ofhuman Notch, (i.e., that portion of human Notch most homologous toDrosophila ELR 11 and 12 (SEQ ID NO:1)). In an alternative embodiment,anti-Delta antibodies can be used for selection.

In another preferred aspect, PCR is used to amplify the desired sequencein the library, prior to selection. For example, oligonucleotide primersrepresenting part of the adhesive domains encoded by a homologue of thedesired gene can be used as primers in PCR.

The above-methods are not meant to limit the following generaldescription of methods by which clones of human Notch and Delta may beobtained.

Any human cell can potentially serve as the nucleic acid source for themolecular cloning of the Notch and Delta gene. The DNA may be obtainedby standard procedures known in the art from cloned DNA (e.g., a DNA"library"), by chemical synthesis, by cDNA cloning, or by the cloning ofgenomic DNA, or fragments thereof, purified from the desired human cell.(See, for example Maniatis et al., 1982, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Glover,D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U.K. Vol. I, II.) Clones derived from genomic DNA may containregulatory and intron DNA regions in addition to coding regions; clonesderived from cDNA will contain only exon sequences. Whatever the source,the gene should be molecularly cloned into a suitable vector forpropagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene may be accomplished in a number ofways. For example, if an amount of a portion of a Notch or Delta (of anyspecies) gene or its specific RNA, or a fragment thereof e.g., theadhesive domain, is available and can be purified and labeled, thegenerated DNA fragments may be screened by nucleic acid hybridization tothe labeled probe (Benton, W. and Davis, R., 1977, Science 196, 180;Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72,3961). Those DNA fragments with substantial homology to the probe willhybridize. It is also possible to identify the appropriate fragment byrestriction enzyme digestion(s) and comparison of fragment sizes withthose expected according to a known restriction map if such isavailable. Further selection can be carried out on the basis of theproperties of the gene. Alternatively, the presence of the gene may bedetected by assays based on the physical, chemical, or immunologicalproperties of its expressed product. For example, cDNA clones, or DNAclones which hybrid-select the proper mRNAs, can be selected whichproduce a protein that, e.g., has similar or identical electrophoreticmigration, isolectric focusing behavior, proteolytic digestion maps, invitro aggregation activity ("adhesiveness") or antigenic properties asknown for Notch or Delta. If an antibody to Notch or Delta is available,the Notch or Delta protein may be identified by binding of labeledantibody to the putatively Notch or Delta synthesizing clones, in anELISA (enzyme-linked immunosorbent assay)-type procedure.

The Notch or Delta gene can also be identified by mRNA selection bynucleic acid hybridization followed by in vitro translation. In thisprocedure, fragments are used to isolate complementary mRNAs byhybridization. Such DNA fragments may represent available, purifiedNotch or Delta DNA of another species (e.g., Drosophila).Immunoprecipitation analysis or functional assays (e.g., aggregationability in vitro; see examples infra) of the in vitro translationproducts of the isolated products of the isolated mRNAs identifies themRNA and, therefore, the complementary DNA fragments that contain thedesired sequences. In addition, specific mRNAs may be selected byadsorption of polysomes isolated from cells to immobilized antibodiesspecifically directed against Notch or Delta protein. A radiolabelledNotch or Delta cDNA can be synthesized using the selected mRNA (from theadsorbed polysomes) as a template. The radiolabelled mRNA or cDNA maythen be used as a probe to identify the Notch or Delta DNA fragmentsfrom among other genomic DNA fragments.

Alternatives to isolating the Notch or Delta genomic DNA include, butare not limited to, chemically synthesizing the gene sequence itselffrom a known sequence or making cDNA to the mRNA which encodes the Notchor Delta gene. For example, RNA for cDNA cloning of the Notch or Deltagene can be isolated from cells which express Notch or Delta. Othermethods are possible and within the scope of the invention.

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas PBR322 or pUC plasmid derivatives. The insertion into a cloningvector can, for example, be accomplished by ligating the DNA fragmentinto a cloning vector which has complementary cohesive termini. However,if the complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and Notch orDelta gene may be modified by homopolymeric tailing. Recombinantmolecules can be introduced into host cells via transformation,transfection, infection, electroporation, etc., so that many copies ofthe gene sequence are generated.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a "shot gun"approach. Enrichment for the desired gene, for example, by sizefractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated Notch or Delta gene, cDNA,or synthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

The human Notch and Delta sequences provided by the instant inventioninclude those nucleotide sequences encoding substantially the same aminoacid sequences as found in human Notch and in human Delta, and thoseencoded amino acid sequences with functionally equivalent amino acids,all as described supra in Section 5.1 for adhesive portions oftoporythmic proteins.

5.3. IDENTIFICATION OF ADDITIONAL MEMBERS OF THE DELTA/SERRATE FAMILY

A rational search for additional members of the Delta/Serrate genefamily may be carried out using an approach that takes advantage of theexistence of the conserved segments of strong homology between Serrateand Delta (see FIG. 10, SEQ ID NO:3 and NO:4). For example, additionalmembers of this gene family may be identified by selecting, from among adiversity of nucleic acid sequences, those sequences that are homologousto both Serrate and Delta (see FIGS. 13A-13F (SEQ ID NO:5), and FIGS.15A-15B (SEQ ID NO:8)), and further identifying, from among the selectedsequences, those that also contain nucleic acid sequences which arenon-homologous to Serrate and Delta. The term "non-homologous" may beconstrued to mean a region which contains at least about 6 contiguousnucleotides in which at least about two nucleotides differ from Serrateand Delta sequence.

For example, a preferred specific embodiment of the invention providesthe following method. Corresponding to two conserved segments betweenDelta and Serrate, Delta AA 63-73 and Delta AA 195-206 (see FIGS.13A-13F, SEQ ID NO:6), sets of degenerate oligonucleotide probes ofabout 10-20 nucleotides may be synthesized, representing all of thepossible coding sequences for the amino acids found in either Delta andSerrate for about three to seven contiguous codons. In anotherembodiment, oligonucleotides may be obtained corresponding to parts ofthe four highly conserved regions between Delta and Serrate shown inFIGS. 15A-15B (SEQ ID NO:8 and NO:9), i.e., that represented by SerrateAA 124-134, 149-158, 214-219, and 250-259. The syntheticoligonucleotides may be utilized as primers to amplify by PCR sequencesfrom a source (RNA or DNA) of potential interest. (PCR can be carriedout, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taqpolymerase (Gene Amp™)). This might include mRNA or cDNA or genomic DNAfrom any eukaryotic species that could express a polypeptide closelyrelated to Serrate and Delta. By carrying out the PCR reactions, it maybe possible to detect a gene or gene product sharing the above-notedsegments of conserved sequence between Serrate and Delta. If one choosesto synthesize several different degenerate primers, it may still bepossible to carry out a complete search with a reasonably small numberof PCR reactions. It is also possible to vary the stringency ofhybridization conditions used in priming the PCR reactions, to allow forgreater or lesser degrees of nucleotide sequence similarity between theunknown gene and Serrate or Delta. If a segment of a previously unknownmember of the Serrate/Delta gene family is amplified successfully, thatsegment may be molecularly cloned and sequenced, and utilized as a probeto isolate a complete cDNA or genomic clone. This, in turn, will permitthe determination of the unknown gene's complete nucleotide sequence,the analysis of its expression, and the production of its proteinproduct for functional analysis. In this fashion, additional genesencoding "adhesive" proteins may be identified.

In addition, the present invention provides for the use of theSerrate/Delta sequence homologies in the design of novel recombinantmolecules which are members of the Serrate/Delta gene family but whichmay not occur in nature. For example, and not by way of limitation, arecombinant molecule can be constructed according to the invention,comprising portions of both Serrate and Delta genes. Such a moleculecould exhibit properties associated with both Serrate and Delta andportray a novel profile of biological activities, including agonists aswell as antagonists. The primary sequence of Serrate and Delta may alsobe used to predict tertiary structure of the molecules using computersimulation (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78,3824-3828); Serrate/Delta chimeric recombinant genes could be designedin light of correlations between tertiary structure and biologicalfunction. Likewise, chimeric genes comprising portions of any one ormore members of the toporythmic gene family (e.g., Notch) may beconstructed.

5.4. THE EXPRESSION OF TOPORYTHMIC GENES

The nucleotide sequence coding for an adhesive fragment of a toporythmicprotein (preferably, Notch, Serrate, or Delta), or an adhesive analog orderivative thereof, or human Notch or Delta or a functionally activefragment or derivative thereof, can be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted protein-codingsequence. The necessary transcriptional and translational signals canalso be supplied by the native toporythmic gene and/or its flankingregions. A variety of host-vector systems may be utilized to express theprotein-coding sequence. These include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.In a specific embodiment, the adhesive portion of the Notch gene, e.g.,that encoding EGF-like repeats 11 and 12, is expressed. In anotherembodiment, the adhesive portion of the Delta gene, e.g., that encodingamino acids 1-230, is expressed. In other specific embodiments, thehuman Notch or human Delta gene is expressed, or a sequence encoding afunctionally active portion of human Notch or Delta. In yet anotherembodiment, the adhesive portion of the Serrate gene is expressed.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequence encoding a toporythmic protein orpeptide fragment may be regulated by a second nucleic acid sequence sothat the toporythmic protein or peptide is expressed in a hosttransformed with the recombinant DNA molecule. For example, expressionof a toporythmic protein may be controlled by any promoter/enhancerelement known in the art. Promoters which may be used to controltoporythmic gene expression include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290, 304-310),the promoter contained in the 3' long terminal repeat of Rous sarcomavirus (Yamamoto, et al., 1980, Cell 22, 787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78,1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296, 39-42); prokaryotic expressionvectors such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75, 3727-3731), or the tac promoter(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 21-25); seealso "Useful proteins from recombinant bacteria" in Scientific American,1980, 242, 74-94; plant expression vectors comprising the nopalinesynthetase promoter region (Herrera-Estrella et al., Nature 303,209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner, etal., 1981, Nucl. Acids Res. 9, 2871), and the promoter of thephotosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrellaet al., 1984, Nature 310, 115-120); promoter elements from yeast orother fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase)promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatasepromoter, and the following animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals: elastase I gene control region which is active in pancreaticacinar cells (Swift et al., 1984, Cell 38, 639-646; Ornitz et al., 1986,Cold Spring Harbor Symp. Quant. Biol. 50, 399-409; MacDonald, 1987,Hepatology 7, 425-515); insulin gene control region which is active inpancreatic beta cells (Hanahan, 1985, Nature 315, 115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38, 647-658; Adames et al., 1985, Nature318, 533-538; Alexander et al., 1987, Mol. Cell. Biol. 7, 1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45, 485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1, 268-276), alpha-fetoprotein gene controlregion which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5, 1639-1648; Hammer et al., 1987, Science 235, 53-58; alpha1-antitrypsin gene control region which is active in the liver (Kelseyet al., 1987, Genes and Devel. 1, 161-171), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315, 338-340; Kollias et al., 1986, Cell 46, 89-94; myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987, Cell 48, 703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314, 283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234, 1372-1378).

Expression vectors containing toporythmic gene inserts can be identifiedby three general approaches: (a) nucleic acid hybridization, (b)presence or absence of "marker" gene functions, and (c) expression ofinserted sequences. In the first approach, the presence of a foreigngene inserted in an expression vector can be detected by nucleic acidhybridization using probes comprising sequences that are homologous toan inserted toporythmic gene. In the second approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain "marker" gene functions (e.g., thymidinekinase activity, resistance to antibiotics, transformation phenotype,occlusion body formation in baculovirus, etc.) caused by the insertionof foreign genes in the vector. For example, if the toporythmic gene isinserted within the marker gene sequence of the vector, recombinantscontaining the toporythmic insert can be identified by the absence ofthe marker gene function. In the third approach, recombinant expressionvectors can be identified by assaying the foreign gene product expressedby the recombinant. Such assays can be based, for example, on thephysical or functional properties of the toporythmic gene product invitro assay systems, e.g., aggregation (adhesive) ability (see Sections6-8, infra).

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered toporythmic protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, cleavage) of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.For example, expression in a bacterial system can be used to produce anunglycosylated core protein product. Expression in yeast will produce aglycosylated product. Expression in mammalian cells can be used toensure "native" glycosylation of a heterologous mammalian toporythmicprotein. Furthermore, different vector/host expression systems mayeffect processing reactions such as proteolytic cleavages to differentextents.

In other specific embodiments, the adhesive toporythmic protein,fragment, analog, or derivative may be expressed as a fusion, orchimeric protein product (comprising the protein, fragment, analog, orderivative joined to a heterologous protein sequence). Such a chimericproduct can be made by ligating the appropriate nucleic acid sequencesencoding the desired amino acid sequences to each other by methods knownin the art, in the proper coding frame, and expressing the chimericproduct by methods commonly known in the art. Alternatively, such achimeric product may be made by protein synthetic techniques, e.g., byuse of a peptide synthesizer.

Both cDNA and genomic sequences can be cloned and expressed.

In other embodiments, a human Notch cDNA sequence may be chromosomallyintegrated and expressed. Homologous recombination procedures known inthe art may be used.

5.4.1. IDENTIFICATION AND PURIFICATION OF THE EXPRESSED GENE PRODUCT

Once a recombinant which expresses the toporythmic gene sequence isidentified, the gene product may be analyzed. This can be achieved byassays based on the physical or functional properties of the product,including radioactive labelling of the product followed by analysis bygel electrophoresis.

Once the toporythmic protein is identified, it may be isolated andpurified by standard methods including chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. The functional properties may be evaluatedusing any suitable assay, including, but not limited to, aggregationassays (see Sections 6-8).

5.5. GENERATION OF ANTIBODIES TO TOPORYTHMIC PROTEINS AND ADHESIVESEQUENCES THEREOF

According to the invention, toporythmic protein fragments or analogs orderivatives thereof which mediate homotypic or heterotypic binding, orhuman Notch or human Delta proteins or fragments thereof, may be used asan immunogen to generate anti-toporythmic protein antibodies. Suchantibodies can be polyclonal or monoclonal. In a specific embodiment,antibodies specific to EGF-like repeats 11 and 12 of Notch may beprepared. In other embodiments, antibodies reactive with the "adhesiveportion" of Delta can be generated. One example of such antibodies mayprevent aggregation in an in vitro assay. In another embodiment,antibodies specific to human Notch are produced.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to a toporythmic protein or peptide. In aparticular embodiment, rabbit polyclonal antibodies to an epitope of thehuman Notch protein encoded by a sequence depicted in FIGS. 19A-19C,20A-20D, 21A-21B or 22A-22D (SEQ ID NO:13 through NO:25), or asubsequence thereof, can be obtained. For the production of antibody,various host animals can be immunized by injection with the nativetoporythmic protein, or a synthetic version, or fragment thereof,including but not limited to rabbits, mice, rats, etc. Various adjuvantsmay be used to increase the immunological response, depending on thehost species, and including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhold limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a toporythmicprotein sequence, any technique which provides for the production ofantibody molecules by continuous cell lines in culture may be used. Forexample, the hybridoma technique originally developed by Kohler andMilstein (1975, Nature 256, 495-497), as well as the trioma technique,the human B-cell hybridoma technique (Kozbor et al., 1983, ImmunologyToday 4, 72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab')₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab' fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragment, andthe Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodieswhich recognize the adhesive domain of a toporythmic protein, one mayassay generated hybridomas for a product which binds to a proteinfragment containing such domain. For selection of an antibody specificto human Notch, one can select on the basis of positive binding to humanNotch and a lack of binding to Drosophila Notch.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the protein sequences ofthe invention. For example, various immunoassays known in the art can beused, including but not limited to competitive and non-competitive assaysystems using techniques such as radioimmiunoassays, ELISA (enzymelinked immunosorbent assay), "sandwich" immunoassays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, fluorescent immunoassays, protein A immunoassays,and immunoelectrophoresis assays, to name but a few.

5.6. DELIVERY OF AGENTS INTO NOTCH-EXPRESSING CELLS

The invention also provides methods for delivery of agents intoNotch-expressing cells. As discussed in Section 8 infra, upon binding toa Notch protein on the surface of a Notch-expressing cell, Delta proteinappears to be taken up into the Notch-expressing cell. The inventionthus provides for delivery of agents into a Notch-expressing cell byconjugation of an agent to a Delta protein or an adhesive fragment orderivative thereof capable of binding to Notch, and exposing aNotch-expressing cell to the conjugate, such that the conjugate is takenup by the cell. The conjugated agent can be, but is not limited to, alabel or a biologically active agent. The biologically active agent canbe a therapeutic agent, a toxin, a chemotherapeutic, a growth factor, anenzyme, a hormone, a drug, a nucleic acid, (e.g., antisense DNA or RNA),etc. In one embodiment, the label can be an imaging agent, including butnot limited to heavy metal contrast agents for x-ray imaging, magneticresonance imaging agents, and radioactive nuclides (i.e., isotopes) forradio-imaging. In a preferred aspect, the agent is conjugated to a sitein the amino terminal half of the Delta molecule.

The Delta-agent conjugate can be delivered to the Notch-expressing cellby exposing the Notch-expressing cell to cells expressing theDelta-agent conjugate or exposing the Notch-expressing cell to theDelta-agent conjugate in a solution, suspension, or other carrier. Wheredelivery is in vivo, the Delta-agent conjugate can be formulated in apharmaceutically acceptable carrier or excipient, to comprise apharmaceutical composition. The pharmaceutically acceptable carrier cancomprise saline, phosphate buffered saline, etc. The Delta-agentconjugate can be formulated as a liquid, tablet, pill, powder, in aslow-release form, in a liposome, etc., and can be administered orally,intravenously, intramuscularly, subcutaneously, intraperitoneally, toname but a few routes, with the preferred choice readily made based onthe knowledge of one skilled in the art.

6. MOLECULAR INTERACTIONS BETWEEN THE PROTEIN PRODUCTS OF THE NEUROGENICLOCI NOTCH AND DELTA, TWO EGF-HOMOLOGOUS GENES IN DROSOPHILA

To examine the possibility of intermolecular association between theproducts of the Notch and Delta genes, we studied the effects of theirexpression on aggregation in Drosophila Schneider's 2 (S2) cells (Fehonet al., 1990, Cell 61, 523-534). We present herein direct evidence ofintermolecular interactions between Notch and Delta, and describe anassay system that will be used in dissecting the components of thisinteraction. We show that normally nonadhesive Drosophila S2 culturedcells that express Notch bind specifically to cells that express Delta,and that this aggregation is calcium dependent. Furthermore, while cellsthat express Notch do not bind to one another, cells that express Deltado bind to one another, suggesting that Notch and Delta can compete forbinding to Delta at the cell surface. We also present evidenceindicating that Notch and Delta form detergent-soluble complexes both incultured cells and embryonic cells, suggesting that Notch and Deltainteract directly at the molecular level in vitro and in vivo. Ouranalyses suggest that Notch and Delta proteins interact at the cellsurface via their extracellular domains.

6.1. EXPERIMENTAL PROCEDURES

6.1.1. EXPRESSION CONSTRUCTS

For the Notch expression construct, the 6 kb HpaI fragment from the 5'end of the Notch coding sequence in MgIIa (Ramos et al., 1989, Genetics123, 337-348) was blunt-end ligated into the metallothionein promotervector pRmHa-3 (Bunch, et al., 1988, Nucl. Acids Res. 16, 1043-1061)after the vector had been cut with EcoRI and the ends were filled withthe Klenow fragment of DNA polymerase I (Maniatis et al., 1982,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory)). A single transformant, incorrectly oriented,was isolated. DNA from this transformant was then digested with SacI,and a resulting 3 kb fragment was isolated that contained the 5' end ofthe Notch coding sequence fused to the polylinker from pRmHa-3. Thisfragment was then ligated into the SacI site of pRmHa-3 in the correctorientation. DNA from this construct was digested with KpnI and XbaI toremove must of the Notch sequence and all of the Adh polyadenylationsignal in pRmHa-3 and ligated to an 11 kb KpnI-XbaI fragment from MgIIacontaining the rest of the Notch coding sequence and 3' sequencesnecessary for polyadenylation. In the resulting construct, designatedpMtNMg, the metallothionein promoter in pRmHa-3 is fused to Notchsequences starting 20 nucleotides upstream of the translation startsite.

For the extracellular Notch construct (ECN1), the CosP479BE Notch cosmid(Ramos et al., 1989, Genetics 123, 337-348), which contains all Notchgenomic sequences necessary for normal Notch function in vivo, waspartially digested with AatII. Fragment ends were made blunt using theexonuclease activity of T4 DNA polymerase (Maniatis et al., 1982,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory)), and the fragments were then redigestedcompletely with StuI. The resulting fragments were separated in a lowmelting temperature agarose gel (SeaPlaque, FMC BioProducts), and thelargest fragment was excised. This fragment was then blunt-end ligatedto itself. This resulted in an internal deletion of the Notch codingsequences from amino acid 1790 to 2625 inclusive (Wharton et al., 1985,Cell 43, 567-581), and a predicted frameshift that produces a novel 59amino acid carboxyl terminus. (The ligated junction of this constructhas not been checked by sequencing.) For the Delta expression construct,the Dl1 cDNA (Kopczynski et al., 1988, Genes Dev. 2, 1723-1735), whichincludes the complete coding capacity for Delta, was inserted into theEcoRI site of pRmHa-3. This construct was called pMTDl1.

6.1.2. ANTIBODY PREPARATION

Hybridoma cell line C17.9C6 was obtained from a mouse immunized with afusion protein based on a 2.1 kb SalI-HindIII fragment that includescoding sequences for most of the intracellular domain of Notch (aminoacids 1791-2504; Wharton et al., 1985, Cell 43, 567-581). The fragmentwas subcloned into pUR289 (Ruther and Muller-Hill, 1983, EMBO J. 2,1791-1794), and then transferred into the pATH 1 expression vector(Dieckmann and Tzagoloff, 1985, J. Biol. Chem. 260, 1513-1520) as aBglII-HindIII fragment. Soluble fusion protein was expressed,precipitated by 25% (NH₄)₂ SO₄, resuspended in 6M urea, and purified bypreparative isoelectric focusing using a Rotofor (Bio-Rad) (for details,see Fehon, 1989, Rotofor Review No. 7, Bulletin 1518, Richmond, Calif.:Bio-Rad Laboratories).

Mouse polyclonal antisera were raised against the extracellular domainof Notch using four BstYl fragments of 0.8 kb (amino acids 237-501:Wharton et al., 1985, Cell 43, 567-581), 1.1 kb (amino acids 501-868),0.99 kb (amino acids 868-1200), and 1.4 kb (amino acids 1465-1935)length, which spanned from the fifth EGF-like repeat across thetransmembrane domain, singly inserted in-frame into the appropriate pGEXexpression vector (Smith and Johnson, 1988, Gene 67, 31-40). Fusionproteins were purified on glutathione-agarose beads (SIGMA). Mouse andrat antisera were precipitated with 50% (NH₄)₂ SO₄ and resuspended inPBS (150 mM NaCl, 14 mM Na₂ HPO₄, 6 mM NaH₂ PO₄) with 0.02% NaN₃.

Hybridoma cell line 201 was obtained from a mouse immunized with afusion protein based on a 0.54 kb ClaI fragment that includes codingsequences from the extracellular domain of Delta (Kopczynski et al.,1988, Genes Dev. 2, 1723-1735) subcloned into the ClaI site within thelacZ gene of pUR 288 (Ruther and Muller-Hill, 1983, EMBO J. 2,1791-1794). This fragment includes sequences extending from the fourththrough the ninth EGF-like repeats in Delta (amino acids 350-529).Fusion protein was prepared by isolation of inclusion bodies (Gilmer etal., 1982, Proc. Natl. Acad. Sci. USA 79, 2152-2156); inclusion bodieswere solubilized in urea (Carroll and Laughon, 1987, in DNA Cloning,Volume III, D. M. Glover, ed. (Oxford: IRL Press), pp. 89-111) beforeuse in immunization.

Rat polyclonal antisera were obtained following immunization withantigen derived from the same fusion protein construct. In this case,fusion protein was prepared by lysis of IPTG-induced cells inSDS-Laemmli buffer (Carroll and Laughon, 1987, in DNA Cloning, VolumeIII, D. M. Glover, ed. (Oxford: IRL Press), pp. 89-111), separation ofproteins by SDS-PAGE, excision of the appropriate band from the gel, andelectroelution of antigen from the gel slice for use in immunization(Harlow and Lane, 1988, Antibodies: A Laboratory Manual (Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory)).

6.1.3. CELL CULTURE AND TRANSFECTION

The S2 cell line (Schneider, 1972, J. Embryol. Exp. Morph. 27, 353-365)was grown in M3 medium (prepared by Hazleton Co.) supplemented with 2.5mg/ml Bacto-Peptone (Difco), 1 mg/ml TC Yeastolate (Difco), 11%heat-inactivated fetal calf serum (FCS) (Hyclone), and 100 U/mlpenicillin-100 μg/ml streptomycin-0.25 μg/ml fungizone (Hazleton). Cellsgrowing in log phase at ˜2×10⁶ cells/ml were transfected with 20 μg ofDNA-calcium phosphate coprecipitate in 1 ml per 5 ml of culture aspreviously described (Wigler et al., 1979, Proc. Natl. Acad. Sci. USA78, 1373-1376), with the exception that BES buffer (SIGMA) was used inplace of HEPES buffer (Chen and Okayama, 1987, Mol. Cell. Biol. 7,2745-2752). After 16-18 hr, cells were transferred to conical centrifugetubes, pelleted in a clinical centrifuge at full speed for 30 seconds,rinsed once with 1/4 volume of fresh complete medium, resuspended intheir original volume of complete medium, and returned to the originalflask. Transfected cells were then allowed to recover for 24 hr beforeinduction.

6.1.4. AGGREGATION ASSAYS

Expression of the Notch and Delta metallothionein constructs was inducedby the addition of CuSO₄ to 0.7 mM. Cells transfected with the ECN1construct were treated similarly. Two types of aggregation assays wereused. In the first assay, a total of 3 ml of cells (5-10×10⁶ cells/ml)was placed in a 25 ml Erlenmeyer flask and rotated at 40-50 rpm on arotary shaker for 24-48 hr at room temperature. For these experiments,cells were mixed 1-4 hr after induction began and induction wascontinued throughout the aggregation period. In the second assay, ˜0.6ml of cells were placed in a 0.6 ml Eppendorf tube (leaving a smallbubble) after an overnight induction (12-16 hr) at room temperature androcked gently for 1-2 hr at 4° C. The antibody inhibition and Ca²⁺dependence experiments were performed using the latter assay. For Ca²⁺dependence experiments, cells were first collected and rinsed inbalanced saline solution (BSS) with 11% FCS (BSS-FCS; FCS was dialyzedagainst 0.9% NaCl, 5 mM Tris pH 7.5!) or in Ca²⁺ free BSS-FCS containing10 mM EGTA (Snow et al., 1989, Cell 59, 313-323) and then resuspended inthe same medium at the original volume. For the antibody inhibitionexperiments, Notch-transfected cells were collected and rinsed in M3medium and then treated before aggregation in M3 medium for 1 hr at 4°C. with a 1:250 dilution of immune or preimmune sera from each of thefour mice immunized with fusion proteins containing segments from theextracellular domain of Notch (see Antibody Preparation above).

6.1.5. IMMUNOFLUORESCENCE

Cells were collected by centrifugation (3000 rpm for 20 seconds in anEppendorf microcentrifuge) and fixed in 0.6 ml Eppendorf tubes with 0.5ml of freshly made 2% paraformaldehyde in PBS for 10 min at roomtemperature. After fixing, cells were collected by centrifugation,rinsed twice in PBS, and stained for 1 hr in primary antibody in PBSwith 0.1% saponin (SIGMA) and 1% normal goat serum (Pocono Rabbit Farm,Canadensis, Pa.). Monoclonal antibody supernatants were diluted 1:10 andmouse or rat sera were diluted 1:1000 for this step. Cells were thenrinsed once in PBS and stained for 1 hr in specific secondary antibodies(double-labeling grade goat anti-mouse and goat anti-rat, JacksonImmunoresearch) in PBS-saponin-normal goat serum. After this incubation,cells were rinsed twice in PBS and mounted on slides in 90% glycerol,10% 1M Tris (pH 8.0), and 0.5% n-propyl gallate. Cells were viewed underepifluorescence on a Leitz Orthoplan 2 microscope.

Confocal micrographs were taken using the Bio-Rad MRC 500 systemconnected to a Zeiss Axiovert compound microscope. Images were collectedusing the BHS and GHS filter sets, aligned using the ALIGN program, andmerged using MERGE. Fluorescent bleed-through from the green into thered channel was reduced using the BLEED program (all software providedby Bio-Rad). Photographs were obtained directly from the computermonitor using Kodak Ektar 125 film.

6.1.6. CELL LYSATES, IMMUNOPRECIPITATIONS, AND WESTERN BLOTS

Nondenaturing detergent lysates of tissue culture and wild-type Canton-Sembryos were prepared on ice in ˜10 cell vol of lysis buffer (300 mMNaCl, 50 mM Tris pH 8.0!, 0.5% NP-40, 0.5% deoxycholate, 1 mM CaCl₂, 1mM MgCl₂) with 1 mM phenylmethysulfonyl fluoride (PMSF) and diisopropylfluorophosphate diluted 1:2500 as protease inhibitors. Lysates weresequentially triturated using 18G, 21G, and 25G needles attached to 1 cctuberculin syringes and then centrifuged at full speed in a microfuge 10min at 4° C. to remove insoluble material. Immunoprecipitation wasperformed by adding ˜1 μg of antibody (1-2 μl of polyclonal antiserum)to 250-500 μl of cell lysate and incubating for 1 hr at 4° C. withagitation. To this mixture, 15 μg of goat anti-mouse antibodies (JacksonImmunoresearch; these antibodies recognize both mouse and rat IgG) wereadded and allowed to incubate for 1 hr at 4° C. with agitation. This wasfollowed by the addition of 100 μl of fixed Staphylococcus aureus (StaphA) bacteria (Zysorbin, Zymed; resuspended according to manufacturer'sinstructions), which had been collected, washed five times in lysisbuffer, and incubated for another hour. Staph A-antibody complexes werethen pelleted by centrifugation and washed three times in lysis bufferfollowed by two 15 min washes in lysis buffer. After being transferredto a new tube, precipitated material was suspended in 50 μl of SDS-PAGEsample buffer, boiled immediately for 10 min, run on 3%-15% gradientgels, blotted to nitrocellulose, and detected using monoclonalantibodies and HRP-conjugated goat anti-mouse secondary antibodies aspreviously described (Johansen et al., 1989, J. Cell Biol. 109,2427-2440). For total cellular protein samples used on Western blots(FIGS. 2A-2B), cells were collected by centrifugation, lysed in 10 cellvol of sample buffer that contained 1 mM PMSF, and boiled immediately.

6.2. RESULTS

6.2.1. THE EXPRESSION OF NOTCH AND DELTA IN CULTURED CELLS

To detect interactions between Notch and Delta, we examined the behaviorof cells expressing these proteins on their surfaces using anaggregation assay. We chose the S2 cell line (Schneider, 1972, J.Embryol. Exp. Morph. 27, 353-365) for these studies for several reasons.First, these cells are relatively nonadhesive, grow in suspension, andhave been used previously in a similar assay to study fasciclin IIIfunction (Snow et al., 1989, Cell 59, 313-323). Second, they are readilytransfectable, and an inducible metallothionein promoter vector that hasbeen designed for expression of exogenous genes in Drosophila culturedcells is available (Bunch et al., 1988, Nucl. Acids Res. 16, 1043-1061).Third, S2 cells express an aberrant Notch message and no detectableNotch due to a rearrangement of the 5' end of the Notch coding sequence(see below). These cells also express no detectable Delta (see below).

Schematic drawings of the constructs used are shown in FIG. 1 (seeExperimental Procedures, Section 6.1, for details). To express Notch incultured cells, the Notch minigene MGlla, described in Ramos et al.(1989, Genetics 123, 337-348) was inserted into the metallothioneinpromoter vector pRmHa-3 (Bunch et al., 1988, Nucl. Acids Res. 16,1043-1061). The Delta expression construct was made by inserting Dl1cDNA, which contains the entire coding sequence for Delta from the majorembryonic Delta transcript (5.4Z; Kopczynski et al., 1988, Genes Dev. 2,1723-1735), into the same vector. A third construct, designated ECN1 for"extracellular Notch 1", contains the 5' Notch promoter region and 3'Notch polyadenylation signal together with coding capacity for theextracellular and transmembrane regions of the Notch gene from genomicsequences, but lacks coding sequences for 835 amino acids of the ˜1000amino acid intracellular domain. In addition, due to a predictedframeshift, the remaining 78 carboxy-terminal amino acid residues arereplaced by a novel 59 amino acid carboxyterminal tail (see ExperimentalProcedures).

For all of the experiments described in this paper, expressionconstructs were transfected into S2 cells and expressed transientlyrather than in stable transformants. Expressing cells typically composed1%-5% of the total cell population, as judged by immunofluorescentstaining (data not shown). A Western blot of proteins expressed aftertransfection is shown in FIGS. 2A-2B. Nontransfected cells do notexpress detectable levels of Notch or Delta. However, aftertransfection, proteins of the predicted apparent molecular weights arereadily detectable using monoclonal antibodies specific for each ofthese proteins, respectively. In the case of Notch, multiple bands wereapparent in transfected cells below the ˜300 kd full-length product. Wedo not yet know whether these bands represent degradation of Notchduring sample preparation or perhaps synthesis or processingintermediates of Notch that are present within cells, but weconsistently detect them in samples from transfected cells and fromembryos. In addition, we performed immunofluorescent staining of livetransfected cells with antibodies specific for the extracellular domainsof each protein to test for cell surface expression of these proteins.In each case we found surface staining as expected for a surfaceantigen. Taken together, these results clearly show that the Notch andDelta constructs support expression of proteins of the expected sizesand subcellular localization.

6.2.2. CELLS THAT EXPRESS NOTCH AND DELTA AGGREGATE

To test the prediction that Notch and Delta interact, we designed asimple aggregation assay to detect these interactions between proteinsexpressed on the surface of S2 cells. We reasoned that if Notch andDelta are able to form stable heterotypic complexes at the cell surface,then cells that express these proteins might bind to one another andform aggregates under appropriate conditions. A similar assay system hasrecently been described for the fasciclin III protein (Snow et al.,1989, Cell 59, 313-323).

S2 cells in log phase growth were separately transfected with either theNotch or Delta metallothionein promoter construct. After induction withCuSO₄, transfected cells were mixed in equal numbers and allowed toaggregate overnight at room temperature (for details, see ExperimentalProcedures, Section 6.1). Alternatively, in some experiments intended toreduce metabolic activity, cells were mixed gently at 4° C. for 1-2 hr.To determine whether aggregates had formed, cells were processed forimmunofluorescence microscopy using antibodies specific for each geneproduct and differently labeled fluorescent secondary antibodies. Aspreviously mentioned, expressing cells usually constituted less than 5%of the total cell population because we used transient rather thanstable transformants. The remaining cells either did not express a givenprotein or expressed at levels too low for detection byimmunofluorescence microscopy. As controls, we performed aggregationswith only a single type of transfected cell.

FIGS. 3A--3I shows representative photomicrographs from aggregationexperiments, and Table I presents the results in numerical form. As isapparent from FIG. 3C and Table I, while Notch-expressing (Notch⁺) cellsalone do not form aggregates in our assay, Delta-expressing (Delta⁺)cells do.

                                      TABLE I    __________________________________________________________________________    PERCENTAGES OF NOTCH.sup.+  AND DELTA.sup.+  CELLS IN AGGREGATES.sup.a           Notch.sup.+                     Delta.sup.+           Control               Aggregated                     Control                         Aggregated                               Notch.sup.+ -Delta           Cells.sup.b               Cells.sup.c                     Cells.sup.b                         Cells.sup.c                               Overall.sup.d                                   N Cells.sup.e                                       D1 Cells.sup.f    __________________________________________________________________________    Experiment 1           0   0     19  37    32  26  42    Experiment 2           --  1     --  40    54  47  79    Experiment 3           0   --    12  --    43  42  44    Experiment 4           5   5     20  --    47  41  59    Experiment 5.sup.gh           --  2     --  48    71  66  82    Experiment 6.sup.h           0   0     13  61    63  60  73    __________________________________________________________________________     .sup.a Aggregates defined as clusters of four or more expressing cells.     For all values, at least 100 expressing cell units (single cells or cell     clusters) were scored. Notch.sup.+, Notchexpressing; Delta.sup.+, Delta     expressing.     .sup.b Control cells taken directly from transfection flasks without     incubation in the aggregation assay.     .sup.c Control cells after incubation in the aggregation assay.     .sup.d Combined aggregation data for both Notch.sup.+  and Delta.sup.+     cells in Notch.sup.+ -Delta.sup.+ aggregates.     .sup.e Aggregation data for Notch.sup.+  cells in Notch.sup.+ -Delta.sup.      aggregates.     .sup.f Aggregation data for Delta.sup.+  cells in Notch.sup.+ -Delta.sup.      aggregates.     .sup.g Cells from this experiment from same transfection as Experiment 4.     .sup.h Data from 48 hr aggregation experiments. All other data are from 2     hr aggregation experiments.

The tendency for Delta⁺ cells to aggregate was apparent even innonaggregated control samples (Table I), where cell clusters of 4-8cells that probably arose from adherence between mitotic sister cellscommonly occurred. However, clusters were more common after incubationunder aggregation conditions (e.g., 19% of Delta⁺ cells in aggregatesbefore incubation vs. 37% of Delta⁺ cells in aggregates afterincubation; Experiment 1 in Table I), indicating that Delta⁺ cells areable to form stable contacts with one another in this assay. It isimportant to note that while nonstaining cells constituted over 90% ofthe cells in our transient transfections, we never found them withinaggregates. On rare occasions, nonstaining cells were found at the edgeof an aggregate. Due to the common occurrence of weakly staining cellsat the edges of aggregates, it is likely that these apparentlynonexpressing cells were transfected but expressed levels of Deltainsufficient to be detected by immunofluorescence.

In remarkable contrast to control experiments with Notch⁺ cells alone,aggregation of mixtures of Notch⁺ and Delta⁺ cells resulted in theformation of clusters of up to 20 or more cells (FIGS. 3D-3H, Table I).As Table I shows, the fraction of expressing cells found in clusters offour or more stained cells after 24 hr of aggregation ranged from32%-54% in mixtures of Notch⁺ and Delta⁺ cells. This range was similarto that seen for Delta⁺ cells alone (37%-40%) but very different fromthat for Notch⁺ cells alone (only 0%-5%). Although a few clusters thatconsisted only of Delta⁺ cells were found, Notch⁺ cells were never foundin clusters of greater than four to five cells unless Delta⁺ cells werealso present. Again, all cells within these clusters expressed eitherNotch or Delta, even though transfected cells composed only a smallfraction of the total cell population. At 48 hr (Table I, experiments 5and 6), the degree of aggregation appeared higher (63%-71%), suggestingthat aggregation had not yet reached a maximum after 24 hr under theseconditions. Also, cells cotransfected with Notch and Delta constructs(so that all transfected cells express both proteins) aggregated in asimilar fashion under the same experimental conditions.

These results indicate that the aggregation observed in theseexperiments requires the expression of Notch and Delta and is not due tothe fortuitous expression of another interacting protein innontransfected S2 cells. We further tested the specificity of thisinteraction by diluting Notch⁺ and Delta⁺ cells 10-fold withnontransfected S2 cells and allowing them to aggregate for 24 hr at roomtemperature. In this experiment, 39% of the expressing cells were foundin aggregates with other expressing cells, although they composed lessthan 0.1% of the total cell population. Not surprisingly, however, theseaggregates were smaller on average than those found in standardaggregation experiments. In addition, to control for the possibilitythat Notch⁺ cells are nonspecifically recruited into the Delta⁺aggregates because they overexpress a single type of protein on the cellsurface, we mixed Delta⁺ cells with cells that expressed neuroglian, atransmembrane cell-surface protein (Bieber et al., 1989, Cell 59,447-460), under the control of the metallothionein promoter (thismetallothionein-neuroglian construct was kindly provided by A. Bieberand C. Goodman). We observed no tendency for neuroglian⁺ cells to adhereto Delta⁺ aggregates, indicating that Notch-Delta aggregation is notmerely the result of high levels of protein expression on the cellsurface.

We also tested directly for Notch involvement in the aggregation processby examining the effect of a mixture of polyclonal antisera directedagainst fusion proteins that spanned almost the entire extracellulardomain of Notch on aggregation (see Experimental Procedures, Section6.1). To minimize artifacts that might arise due to a metabolic responseto patching of surface antigens, antibody treatment and the aggregationassay were performed at 4° C. in these experiments. Notch⁺ cells wereincubated with either preimmune or immune mouse sera for 1 hr, Delta⁺cells were added, and aggregation was performed for 1-2 hr. While Notch⁺cells pretreated with preimmune sera aggregated with Delta⁺ cells (inone of three experiments, 23% of the Notch⁺ cells were in Notch⁺ -Delta⁺cell aggregates), those treated with immune sera did not (only 2% ofNotch⁺ cells were in aggregates). This result suggests that theextracellular domain of Notch is required for Notch⁺ -Delta⁺ cellaggregation, although we cannot rule out the possibility that thereduced aggregation was due to inhibitory steric or membrane structureeffects resulting from exposure of Notch⁺ cells to the antiserum.

Three other observations worth noting are apparent in FIGS. 3A--3I.First, while Delta was almost always apparent only at the cell surface(FIGS. 3B and 3C), Notch staining was always apparent both at the cellsurface and intracellularly, frequently associated with vesicularstructures (FIG. 3A). Second, we consistently noted a morphologicaldifference between Delta⁺ and Notch⁺ cells in mixed aggregates that wereincubated overnight. Delta⁺ cells often had long extensions thatcompletely surrounded adjacent Notch⁺ cells, while Notch⁺ cells werealmost always rounded in appearance without noticeable cytoplasmicextensions (FIG. 3G). Third, Notch and Delta often appeared to gatherwithin regions of contact between Notch⁺ and Delta⁺ cells, producing asharp band of immunofluorescent staining (FIGS. 3D-3F). These bands werereadily visible in optical sections viewed on the confocal microscope(FIG. 3H), indicating that they were not merely due to a whole-mountartifact. We also observed that these bands formed rapidly (within 2 hrof mixing cells) and at 4° C., indicating that their formation probablydid not depend upon cellular metabolism. These observations would beexpected if, within regions of cell contact, Notch and Delta bind to oneanother and therefore become immobilized. This pattern of expression isalso consistent with that observed for other proteins that mediate cellaggregation (Takeichi, 1988, Development 102, 639-655; Snow et al.,1989, Cell 59, 313-323).

6.2.3. NOTCH-DELTA-MEDIATED AGGREGATION IS CALCIUM DEPENDENT

Previous studies have suggested that EGF-like repeats that contain aparticular consensus sequence may serve as calcium (Ca²⁺) bindingdomains (Morita et al., 1984, J. Biol. Chem. 259, 5698-5704; Sugo etal., 1984, J. Biol. Chem. 259, 5705-5710; Rees et al., 1988, EMBO J. 7,2053-2061; Handford et al., 1990, EMBO J. 9, 475-480). For at least twoof these proteins, C and C1, Ca²⁺ binding has further been shown to be anecessary component of their interactions with other proteins (Villierset al., 1980, FEBS Lett. 117, 289-294; Esmon et al., 1983, J. Biol.Chem. 258, 5548-5553; Johnson, et al., 1983, J. Biol. Chem. 258,5554-5560). Many of the EGF-homologous repeats within Notch and most ofthose within Delta contain the necessary consensus sequence for Ca²⁺binding (Rees et al., 1988, EMBO J. 7, 2053-2061; Stenflo et al., 1987,Proc. Natl. Acad. Sci. USA 84, 368-372; Kopczynski et al., 1988, GenesDev. 2, 1723-1735; Handford et al., 1990, EMBO J. 9, 475-480), althoughit has not yet been determined whether or not these proteins do bindcalcium. We therefore tested the ability of expressing cells toaggregate in the presence or absence of Ca²⁺ ions to determine whetherthere is a Ca²⁺ ion requirement for Notch-Delta aggregation. To minimizepossible nonspecific effects due to metabolic responses to the removalof Ca²⁺, these experiments were performed at 4° C. Control mixtures ofNotch⁺ and Delta⁺ cells incubated under aggregation conditions in Ca²⁺-containing medium at 4° C. readily formed aggregates (an average of34%±13%, mean±SD, n=3; Table II). In contrast, cells mixed in mediumthat lacked Ca²⁺ ions and contained EGTA formed few aggregates (5%±5%).These results clearly demonstrate a dependence of Notch-Delta-mediatedaggregation on exogenous Ca²⁺ and are in marked contrast to thoserecently published for the Drosophila fasciclin III and fasciclin Iproteins in S2 cells (Snow et al., 1989, Cell 59, 313-323; Elkins etal., 1990, J. Cell Biol. 110, 1825-1832), which detected no effect ofCa²⁺ ion removal on aggregation mediated by either protein.

                  TABLE II    ______________________________________    EFFECT OF EXOGENOUS Ca.sup.2+  ON    NOTCH.sup.+ -DELTA.sup.+  AGGREGATION.sup.a           Without Ca2+ Ions                         With Ca.sup.2+  Ions           Over- N       D1      Over- N     D1           all.sup.b                 Cells.sup.c                         Cells.sup.d                                 all.sup.b                                       Cells.sup.c                                             Cells.sup.d    ______________________________________    Experiment 1             4       2       5     28    28    27    Experiment 2             12      0       13    53    63    50    Experiment 3             0       0       0     22    28    17    ______________________________________     .sup.a Data presented as percentage of expressing cells found in     aggregates (as in Table I).     .sup.b Combined aggregation data for both Notch.sup.+  and Delta.sup.+     cells.     .sup.c Aggregation data for Notch.sup.+  cells in Notch.sup.+ -Delta.sup.      aggregates.     .sup.d Aggregation data for Delta.sup.+  cells in Notch.sup.+ -Delta.sup.      aggregates.

6.2.4. NOTCH AND DELTA INTERACT WITHIN A SINGLE CELL

We asked whether Notch and Delta are associated within the membrane ofone cell that expresses both proteins by examining the distributions ofNotch and Delta in cotransfected cells. As shown in FIGS. 4A and 4B,these two proteins often show very similar distributions at the surfaceof cotransfected cells. To test whether the observed colocalization wascoincidental or represented a stable interaction between Notch andDelta, we treated live cells with an excess of polyclonal anti-Notchantiserum. This treatment resulted in "patching" of Notch on the surfaceof expressing cells into discrete patches as detected byimmunofluorescence. There was a distinct correlation between thedistributions of Notch and Delta on the surfaces of these cells afterthis treatment (FIGS. 4C and 4D), indicating that these proteins areassociated within the membrane. It is important to note that theseexperiments do not address the question of whether this association isdirect or mediated by other components, such as the cytoskeleton. Tocontrol for the possibility that Delta is nonspecifically patched inthis experiment, we cotransfected cells with Notch and with thepreviously mentioned neuroglian construct (A. Bieber and C. Goodman,unpublished data) and patched with anti-Notch antisera. In this casethere was no apparent correlation between Notch and neuroglian.

6.2.5. INTERACTIONS WITH DELTA DO NOT REQUIRE THE INTRACELLULAR DOMAINOF NOTCH

In addition to a large extracellular domain that contains EGF-likerepeats, Notch has a sizeable intracellular (IC) domain of ˜940 aminoacids. The IC domain includes a phosphorylation site (Kidd et al., 1989,Genes Dev. 3, 1113-1129), a putative nucleotide binding domain, apolyglutamine stretch (Wharton et al., 1985, Cell 43, 567-581; Kidd, etal., 1986, Mol. Cell. Biol. 6, 3094-3108), and sequences homologous tothe yeast cdc10 gene, which is involved in cell cycle control in yeast(Breeden and Nasmyth, 1987, Nature 329, 651-654). Given the size andstructural complexity of this domain, we wondered whether it is requiredfor Notch-Delta interactions. We therefore used a variant Notchconstruct from which coding sequences for ⁻ 835 amino acids of the ICdomain, including all of the structural features noted above, had beendeleted (leaving 25 membrane-proximal amino acids and a novel 59 aminoacid carboxyl terminus; see Experimental Procedures and FIG. 1 fordetails). This construct, designated ECN1, was expressed constitutivelyunder control of the normal Notch promoter in transfected cells at alevel lower than that observed for the metallothionein promoterconstructs, but still readily detectable by immunofluorescence.

In aggregation assays, cells that expressed the ECN1 constructconsistently formed aggregates with Delta⁺ cells (31% of ECN1-expressingcells were in aggregates in one of three experiments; see also FIG. 3I),but not with themselves (only 4% in aggregates), just as we observed forcells that expressed intact Notch. We also observed sharp bands of ECN1staining within regions of contact with Delta⁺ cells, again indicating alocalization of ECN1 within regions of contact between cells. To testfor interactions within the membrane, we repeated the surface antigenco-patching experiments using cells cotransfected with the ECN1 andDelta constructs. As observed for intact Notch, we found that when ECN1was patched using polyclonal antisera against the extracellular domainof Notch, ECN1 and Delta colocalized at the cell surface (FIGS. 4E and4F). These results demonstrate that the observed interactions betweenNotch and Delta within the membrane do not require the deleted portionof the IC domain of Notch and are therefore probably mediated by theextracellular domain. However, it is possible that the remainingtransmembrane or IC domain sequences in ECN1 are sufficient to mediateinteractions within a single cell.

6.2.6. NOTCH AND DELTA FORM DETERGENT-SOLUBLE INTERMOLECULAR COMPLEXES

Together, we take the preceding results to indicate molecularinteractions between Notch and Delta present within the same membraneand between these proteins expressed on different cells. As a furthertest for such interactions, we asked whether these proteins wouldcoprecipitate from nondenaturing detergent extracts of cells thatexpress Notch and Delta. If Notch and Delta form a stable intermolecularcomplex either between or within cells, then it should be possible toprecipitate both proteins from cell extracts using specific antiseradirected against one of these proteins. We performed this analysis byimmunoprecipitating Delta with polyclonal antisera fromNP-40/deoxycholate lysates (see Experimental Procedures) of cellscotransfected with the Notch and Delta constructs that had been allowedto aggregate overnight or of 0-24 hr wild-type embryos. We were unableto perform the converse immunoprecipitates because it was not possibleto discern unambiguously a faint Delta band among background Staph Abands. It is important to note that we tested this polyclonal anti-Deltaantiserum for cross-reactivity against Notch in cell lysates (FIG. 5A,lane 1) and by immunofluorescence (e.g., compare FIGS. 3D and 3E) andfound none. After repeated washing to remove nonspecifically adheringproteins, we assayed for coprecipitation of Notch using a monoclonalantibody (MAb C17.9C6) against Notch on Western blots.

As FIGS. 5A-5B shows, we did detect coprecipitation of Notch in Deltaimmunoprecipitates from cotransfected cells and embryos. However,coprecipitating Notch appeared to be present in much smaller quantitiesthan Delta and was therefore difficult to detect. This disparity is mostlikely due to the disruption of Notch-Delta complexes during the lysisand washing steps of the procedure. However, it is also possible thatthis disparity reflects a nonequimolar interaction between Notch andDelta or greatly different affinities of the antisera used to detectthese proteins. The fact that immunoprecipitation of Delta results inthe coprecipitation of Notch constitutes direct evidence that these twoproteins form stable intermolecular complexes in transfected S2 cellsand in embryonic cells.

6.3. DISCUSSION

We have studied interactions between the protein products of two of theneurogenic loci, Notch and Delta, in order to understand their cellularfunctions better. Using an in vitro aggregation assay that employsnormally nonadhesive S2 cells, we showed that cells that express Notchand Delta adhere specifically to one another. The specificity of thisinteraction is apparent from the observation that Notch⁺ -Delta⁺ cellaggregates rarely contained nonexpressing cells, even thoughnonexpressing cells composed the vast majority of the total cellpopulation in these experiments. We propose that this aggregation ismediated by heterotypic binding between the extracellular domains ofNotch and Delta present on the surfaces of expressing cells. Consistentwith this proposal, we find that antisera directed against theextracellular domain of Notch inhibit Notch-Delta-mediated aggregation,and that the ECN1 Notch variant, which lacks almost all of the Notchintracellular domain, can mediate aggregation with cells that expressDelta. We also found that cells that express only Delta aggregate withone another, while those that express only Notch do not. These findingssuggest that Delta can participate in a homotypic interaction whenpresent on apposed cell surfaces but that Notch cannot under our assayconditions.

The proposal that Notch and Delta interact at the cell surface isfurther supported by three lines of evidence. First, we find an intenselocalization of both proteins within regions of contact which Notch⁺ andDelta⁺ cells, implying that Notch and Delta interact directly, even whenexpressed in different cells. Second, Notch and Delta colocalize on thesurface of cells that express both proteins, suggesting that theseproteins can interact within the cell membrane. Third, Notch and Deltacan be coprecipitated from nondenaturing detergent extracts of culturedcells that express both proteins as well as from extracts of embryoniccells. Together, these results strongly support the hypothesis thatNotch and Delta can interact heterotypically when expressed on thesurfaces of either the same or different cells.

The underlying basis for the observed genetic interactions between Notchand Delta and between Notch and mam (Xu et al., 1990, Genes Dev. 4,464-475) may be a dose-sensitive interaction between the proteinsencoded by these genes.

Two lines of evidence suggest that the Notch and Delta proteins functionsimilarly in vitro and in vivo. First, the genetic analyses haveindicated that the stoichiometry of Notch and Delta is crucial for theirfunction in development. Our observations that both Notch-Delta andDelta--Delta associations may occur in vitro imply that Notch and Deltamay compete for binding to Delta. Thus, dose-sensitive geneticinteractions between Notch and Delta may be the result of competitivebinding interactions between their protein products. Second, we wereable to detect Notch-Delta association in lysates of cultured cells andin lysates of Drosophila embryos using immunoprecipitation. Takentogether, these genetic and biochemical analyses suggest that Notch andDelta do associate in vivo in a manner similar to that which we proposeon the basis of our aggregation assays.

Genetic and molecular analyses of Notch have also raised the possibilitythat there may be interactions between individual Notch proteins(Portin, 1975, Genetics 81, 121-133; Kelley et al., 1987, Cell 51,539-548; Artavanis-Tsakonas, 1988, Trends Genet. 4, 95-100). Indeed,Kidd et al. (1989, Genes Dev. 3, 1113-1129) have proposed that thisprotein forms disulfide cross-linked dimers, although this point has notyet been rigorously proven. With or without the formation of covalentcross-links, such interactions could presumably occur either within asingle cell or between cells. However, our find that Notch⁺ cells do notaggregate homotypically suggests that Notch--Notch associations arelikely to occur within a single cell and not between cells.Alternatively, it is possible that homotypic Notch interactions requiregene products that are not expressed in S2 cells.

The Notch-Delta interactions indicated by our analysis are probablymediated by the extracellular domains of these proteins. Aggregationexperiments using the ECN1 construct, from which almost the entireintracellular domain of Notch has been removed or altered by in vitromutagenesis, confirmed this conclusion. Further experiments thatdemonstrate ECN1-Delta associations within the membrane on the basis oftheir ability to co-patch indicated that these interactions are alsolikely to be mediated by the extracellular domains of Notch and Delta,although in this case we cannot exclude possible involvement of thetransmembrane domain or the remaining portion of the Notch intracellulardomain. These results are especially interesting in light of the factthat both Notch and Delta have EGF-like repeats within theirextracellular domains (Wharton et al., 1985, Cell 43, 567-581; Kidd etal., 1986, Mol. Cell Biol. 6, 3094-3108; Vassin et al., 1987, EMBO J. 6,3431-3440; Kopczynski et al., 1988, Genes Dev. 2, 1723-1735).

A second issue of interest regarding EGF domains is the proposal thatthey can serve as Ca²⁺ binding domains when they contain a consensussequence consisting of Asp, Asp/Asn, Asp/Asn, and Tyr/Phe residues atconserved positions within EGF-like repeats (Rees et al., 1988, EMBO J.7, 2053-2061; Handford et al., 1990, EMBO J. 9, 475-480). Comparisonswith a proposed consensus sequence for Ca²⁺ binding have revealed thatsimilar sequences are found within many of the EGF-like repeats of Notch(Rees et al., 1988, EMBO J. 7, 2053-2061) and within most of theEGF-like repeats of Delta (Kopczynski et al., 1988, Genes Dev. 2,1723-1735). Furthermore, sequence analyses of Notch mutations have shownthat certain Ax alleles are associated with changes in amino acidswithin this putative Ca²⁺ binding domain (Kelley et al., 1987, Cell 51,539-548; Hartley et al., 1987, EMBO J. 6, 3407-3417; Rees et al., 1988,EMBO J. 7, 2053-2061). For example, the AxE² mutation, which correlateswith a His to Tyr change in the 29th EGF-like repeat, appears to changethis repeat toward the consensus for Ca²⁺ binding. Conversely, theAx^(9B2) mutation appears to change the 24th EGF-like repeat away fromthis consensus as a result of an Asp to Val change. Thus, the geneticinteractions between Ax alleles and Delta mutations (Xu et al., 1990,Genes Dev., 4, 464-475) raise the possibility that Ca²⁺ ions play a rolein Notch-Delta interactions. Our finding that exogenous Ca²⁺ isnecessary for Notch-Delta-mediated aggregation of transfected S2 cellssupports this contention.

As we have argued (Johansen et al., 1989, J. Cell Biol. 109, 2427-2440;Alton et al., 1989, Dev. Genet. 10, 261-272), on the basis of previousmolecular and genetic analyses one could not predict with any certaintythe cellular function of either Notch or Delta beyond their involvementin cell--cell interactions. However, given the results presented here,it now seems reasonable to suggest that Notch and Delta may function invivo to mediate adhesive interactions between cells. At the same time,it is quite possible that the observed Notch-Delta interactions may notreflect a solely adhesive function and may in addition reflectreceptor-ligand binding interactions that occur in vivo. Indeed, thepresence of a structurally complex 1000 amino acid intracellular domainwithin Notch may be more consistent with a role in signal transductionthan with purely adhesive interactions. Given that Notch may have anadhesive function in concert with Delta, axonal expression of Notch mayplay some role in axon guidance.

7. EGF REPEATS 11 AND 12 OF NOTCH ARE REQUIRED AND SUFFICIENT FORNOTCH-DELTA-MEDIATED AGGREGATION

In this study, we use the same aggregation assay as described in Section6, together with deletion mutants of Notch to identify regions withinthe extracellular domain of Notch necessary for interactions with Delta.We present evidence that the EGF repeats of Notch are directly involvedin this interaction and that only two of the 36 EGF repeats appearnecessary. We demonstrate that these two EGF repeats are sufficient forbinding to Delta and that the calcium dependence of Notch-Delta mediatedaggregation also associates with these two repeats. Finally, the twocorresponding EGF repeats from the Xenopus homolog of Notch also mediateaggregation with Delta, implying that not only has the structure ofNotch been evolutionarily conserved, but also its function. Theseresults suggest that the extracellular domain of Notch is surprisinglymodular, and could potentially bind a variety of proteins in addition toDelta.

7.1. EXPERIMENTAL PROCEDURES

7.1.1. EXPRESSION CONSTRUCTS

The constructs described are all derivatives of the full length Notchexpression construct #1 pMtNMg (see Section 6, supra). All ligationswere performed using DNA fragments cut from low melting temperatureagarose gels (Sea Plaque, FMC BioProducts). The 6 kb EcoRI-XhoI fragmentfrom pMtNMg containing the entire extracellular domain of Notch wasligated into the EcoRI-XhoI sites of the Bluescript vector (Stratagene),and named RI/XBS. All subsequent deletions and insertions of EGF repeatswere performed in this subclone. The Notch sequence containing theEcoRI-XhoI fragment of these RI/XBS derivatives was then mixed with the5.5 kb XhoI-XbaI fragment from pMtNMg containing the intracellulardomain and 3' sequences needed for polyadenylation, and then insertedinto the EcoRI-XbaI site of pRMHa-3 (Bunch et al., 1988, Nucl. AcidsRes. 16, 1043-1061) in a three piece ligation. All subsequent numbersrefer to nucleotide coordinates of the Notch sequence according toWharton et al. (1985, Cell 43, 567-581).

For construct #2 DSph, RI/XBS was digested to completion with SphI andthen recircularized, resulting in a 3.5 kb in-frame deletion fromSphI(996) to SphI(4545).

For construct #3 ΔCla, RI/XBS was digested to completion with ClaI andthen religated, producing a 2.7 kb in-frame deletion from ClaI(1668) toClaI(4407). The ligation junction was checked by double strandsequencing (as described by Xu et al., 1990, Genes Dev. 4, 464-475)using the Sequenase Kit (U.S. Biochemical Corp., Cleveland). We foundthat although the ClaI site at position 4566 exists according to thesequence, it was not recognized under our conditions by the ClaIrestriction enzyme.

For constructs #4-12, RI/XBS was partially digested with ClaI and thenreligated to produce all possible combinations of in-frame deletions:construct #4 ΔEGF7-17 removed the sequence between ClaI(1668) andClaI(2820); Construct #5 ΔEGF9-26 removed the sequence betweenClaI(1905) and ClaI(3855); construct #6 ΔEGF17-31 removed the sequencebetween ClaI(2820) and ClaI(4407); construct #7 ΔEGF7-9 removed thesequence between ClaI(1668) and ClaI(1905); construct #8 ΔEGF9-17removed the sequence between ClaI(1905) and ClaI(2820); construct #9ΔEGF17-26 removed the sequence between ClaI(2820) and ClaI(3855);construct #10 ΔEGF 26-30 removed the sequence between ClaI(3855) andClaI(4407); construct #11 ΔEGF9-30 removed the sequence betweenClaI(1905) and ClaI(4407); construct #12 ΔEGF 7-26 removed the sequencebetween ClaI(1668) and ClaI(3855).

For constructs #13 ΔCla+EGF9-17 and #14 ΔCla+EGF17-26, the ˜0.9 kbfragment between ClaI(1905) and ClaI(2820), and the ˜1.0 kb fragmentbetween ClaI(2820) and ClaI(3855), respectively, were inserted into theunique ClaI site of construct #3 ΔCla.

For construct #16 split, the 11 kb KpnI/XbaI fragment of pMtNMg wasreplaced with the corresponding KpnI/XbaI fragment from a Notch minigeneconstruct containing the split mutation in EGF repeat 14.

For constructs #17-25, synthetic primers for polymerase chain reaction(PCR) were designed to amplify stretches of EGF repeats while breakingthe EGF repeats at the ends of the amplified piece in the same place asthe common ClaI sites just after the third cysteine of the repeat (seeFIG. 7). The PCR products were gel purified as usual and ligated intothe ClaI site of construct #3 ΔCla which was made blunt by filling withthe Klenow fragment of DNA Polymerase I (Maniatis et al., 1990,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). The correct orientation of the inserts wasdetermined by PCR using a sense strand primer within the insert togetherwith an antisense strand primer in EGF repeat 35. All primers were20-mers, and were named with the number of the nucleotide at their 5'end, according to the nucleotide coordinates of the Notch sequence inWharton et al. (1985, Cell 43, 567-581), and S refers to a sense strandprimer while A refers to an antisense strand primer. Construct #16ΔCla+EGF(9-13) used primers S1917 and A2367. Construct #17ΔCla+EGF(11-15) used primers S2141 and A2591. Construct #18ΔCla+EGF(13-17) used primers S2375 and A2819. Construct #19ΔCla+EGF(10-13) used primers S2018 and A2367. Construct #20ΔCla+EGF(11-13) used primers S2141 and A2367. Construct #21 ΔCla+EGF(10-12) used primers S2018 and A2015. Construct #22 ΔCla+EGF(10-11) usedprimers S2018 and A2322. Construct #23 ΔCla+EGF(10-12) used primersS2018 and A2322. Construct #24 ΔCla+EGF(11-12) used primers S2081 andA2322.

For construct #25 ΔEGF, construct R1/XBS was digested to completion withSphI(996) and partially digested with BamHI(5135). The resultingincompatible ends were joined using a synthetic linker designed tocreate a unique ClaI site. This produced an in frame deletion whichremoved all 36 EGF repeats with the exception of the first half ofrepeat 1. For constructs #26-29, the EGF fragments were inserted intothis ClaI site as previously described for the corresponding constructs#13, 16, 19, and 23.

For construct #30 ΔECN, construct R1/XBS was digested to completion withBglI, EcoRI and XhoI. The ˜0.2 kb EcoRI-BglI fragment (722-948) and the˜0.7 kb BglI-XhoI (5873-6627) fragments were ligated with EcoRI-XhoI cutBluescript vector and a synthetic linker designed to create a uniqueClaI site, resulting in an in-frame deletion from BglI(941) toBglI(5873) that removed all 36 EGF repeats except for the first third ofrepeat 1 as well as the 3 Notch/lin-12 repeats. For constructs #31 and32, the EGF fragments were inserted into the unique ClaI site aspreviously described for constructs #19 and 23.

For constructs #33 and 34, PCR primers S1508 and A1859 based on theXenopus Notch sequence (Coffman et al., 1990, Science 249, 1438-1441;numbers refer to nucleotide coordinates used in this paper), were usedto amplify EGF repeats 11 and 12 out of a Xenopus stage 17 cDNA library(library was made by D. Melton and kindly provided by M. Danilchek). Thefragment was ligated into construct #3 DCla and sequenced.

7.1.2. CELL CULTURE AND TRANSFECTION

The Drosphila S2 cell line was grown and transfected as described inSection 6, supra. The Delta-expressing stably transformed S2 cell lineL-49-6-7 (kindly established by L. Cherbas) was grown in M3 medium(prepared by Hazleton Co.) supplemented with 11% heat inactivated fetalcalf serum (FCS) (Hyclone), 100 U/ml penicillin-100 μg/mlstreptomycin-0.25 μg/ml fungizone (Hazleton), 2×10⁻⁷ M methotrexate, 0.1mM hypoxanthine, and 0.016 mM thymidine.

7.1.3. AGGREGATION ASSAYS AND IMMUNOFLUORESCENCE

Aggregation assays and Ca⁺⁺ dependence experiments were as describedsupra, Section 6. Cells were stained with the anti-Notch monoclonalantibody 9C6.C17 and anti-Delta rat polyclonal antisera (detailsdescribed in Section 6, supra). Surface expression of Notch constructsin unpermeabilized cells was assayed using rat polyclonal antiseraraised against the 0.8 kb (amino acids 237-501; Wharton et al., 1985,Cell 43, 567-581) BstYI fragment from the extracellular domain of Notch.Cells were viewed under epifluorescence on a Leitz Orthoplan 2microscope.

7.2. RESULTS

7.2.1. EGF REPEATS 11 AND 12 OF NOTCH ARE REQUIRED FOR NOTCH-DELTAMEDIATED AGGREGATION

We have undertaken an extensive deletion analysis of the extracellulardomain of the Notch protein, which we have shown (supra, Section 6) tobe involved in Notch-Delta interactions, to identify the precise domainof Notch mediating these interactions. We tested the ability of cellstransfected with the various deletion constructs to interact with Deltausing the aggregation assay described in Section 6. Briefly, Notchdeletion constructs were transiently transfected into Drosophila S2cells, induced with CuSO₄, and then aggregated overnight at roomtemperature with a small amount of cells from the stably transformedDelta expressing cell line L49-6-7(Cherbas), yielding a populationtypically composed of ˜1% Notch expressing cells and ˜5% Deltaexpressing cells, with the remaining cells expressing neither protein.To assay the degree of aggregation, cells were stained with antiseraspecific to each gene product and examined with immunofluorescentmicroscopy (see experimental procedures for details). Aggregates weredefined as clusters of four or more cells containing both Notch andDelta expressing cells, and the values shown in FIGS. 6A-6B representthe percentage of all Notch expressing cells found in such clusters. Allnumbers reflect the average result from at least two separatetransfection experiments in which at least 100 Notch expressing cellunits (either single cells or clusters) were scored.

Schematic drawings of the constructs tested and results of theaggregation experiments are shown in FIGS. 6A-6B (see ExperimentalProcedures for details). All expression constructs were derivatives ofthe full length Notch expression construct #1 pMtNMg (described inSection 6, supra).

The initial constructs (#2 DSph and #3 ΔCla) deleted large portions ofthe EGF repeats. Their inability to promote Notch-Delta aggregationsuggested that the EGF repeats of Notch were involved in the interactionwith Delta. We took advantage of a series of six in-frame ClaIrestriction sites to further dissect the region between EGF repeats 7and 30. Due to sequence homology between repeats, five of the ClaI sitesoccur in the same relative place within the EGF repeat, just after thethird cysteine, while the sixth site occurs just before the firstcysteine of EGF repeat 31 (FIG. 7). Thus, by performing a partial ClaIdigestion and then religating, we obtained deletions that not onlypreserved the open reading frame of the Notch protein but in additionfrequently maintained the structural integrity and conserved spacing, atleast theoretically, of the three disulfide bonds in the chimeric EGFrepeats produced by the religation (FIGS. 6A-6B, constructs #4-14).Unfortunately, the most 3' ClaI site was resistant to digestion whilethe next most 3' ClaI site broke between EGF repeats 30 and 31.Therefore, when various ClaI digestion fragments were reinserted intothe framework of the complete ClaI digest (construct #3 ΔCla), theoverall structure of the EGF repeats was apparently interrupted at the3' junction.

Several points about this series of constructs are worth noting. First,removal of the ClaI restriction fragment breaking in EGF repeats 9 and17 (construct #8 ΔEGF9-17) abolished aggregation with Delta, whilereinsertion of this piece into construct #3 ΔCla, which lacks EGFrepeats 7-30, restored aggregation to roughly wild type levels(construct #13 ΔCla+EGF9-17), suggesting that EGF repeats 9 through 17contain sequences important for binding to Delta. Second, all constructsin this series (#4-14) were consistent with the binding site mapping toEGF repeats 9 through 17. Expression constructs containing these repeats(#6, 7, 9, 10, 13) promoted Notch-Delta interactions while constructslacking these repeats (#4, 5, 8, 11, 12, 14) did not. To confirm thatinability to aggregate with Delta cells was not simply due to failure ofthe mutagenized Notch protein to reach the cell surface, but actuallyreflected the deletion of the necessary binding site, we tested for cellsurface expression of all constructs by immunofluorescently staininglive transfected cells with antibodies specific to the extracellulardomain of Notch. All constructs failing to mediate Notch-Deltainteractions produced a protein that appeared to be expressed normallyat the cell surface. Third, although the aggregation assay is notquantitative, two constructs which contained EGF repeats 9-17, #9ΔEGF17-26 or most noticeably #10 ΔEGF26-30, aggregated at a seeminglylower level. Cells transfected with constructs #9 ΔEGF17-26 and 10ΔEGF26-30 showed considerably less surface staining than normal,although fixed and permeabilized cells reacted with the same antibodystained normally, indicating we had not simply deleted the epitopesrecognized by the antisera. By comparing the percentage of transfectedcells in either permeabilized or live cell populations, we found thatroughly 50% of transfected cells for construct #9 ΔEGF17-26 and 10% forconstruct #10 ΔEGF26-30 produced detectable protein at the cell surface.Thus these two constructs produced proteins which often failed to reachthe cell surface, perhaps because of misfolding, thereby reducing, butnot abolishing, the ability of transfected cells to aggregate withDelta-expressing cells.

Having mapped the binding site to EGF repeats 9 through 17, we checkedwhether any Notch mutations whose molecular lesion has been determinedmapped to this region. The only such mutation was split, a semidominantNotch allele that correlates with a point mutation in EGF repeat 14(Hartley et al., 1987, EMBO J. 6, 3407-3417; Kelley et al., 1987, Mol.Cell. Biol. 6, 3094-3108). In fact, a genetic screen for second sitemodifiers of split revealed several alleles of Delta, suggesting aspecial relationship between the split allele of Notch, and Delta (Brandand Campus-Ortega, 1990, Roux's Arch. Dev. Biol. 198(5), 275-285). Totest for possible effects of the split mutation on Notch-Delta mediatedaggregation, an 11 kb fragment containing the missense mutationassociated with split was cloned into the Notch expression construct(#15 split). However, aggregation with Delta-expressing cells wasunaffected in this construct suggesting, as was confirmed by subsequentconstructs, that EGF repeat 14 of Notch was not involved in theinteractions with Delta modelled by our tissue culture assay.

Thus, to further map the Delta binding domain within EGF repeats 9-17,we used specific oligonucleotide primers and the PCR technique togenerate several subfragments of this region. To be consistent withconstructs #4-14 which produced proteins that were able to interact withDelta, we designed the primers to splice the EGF repeats just after thethird cysteine, in the same place as the common ClaI site (FIG. 7). Theresulting PCR products were ligated into the ClaI site of construct #3ΔCla. Three overlapping constructs, #16, 17 and 18 were produced, onlyone of which, #16 ΔCla+EGF9-13, when transfected into S2 cells, allowedaggregation with Delta cells. Construct #19 ΔCla+EGF(10-13), which lacksEGF repeat 9, further defined EGF repeats 10-13 as the region necessaryfor Notch-Delta interactions.

Constructs #20-24 represented attempts to break this domain down evenfurther using the same PCR strategy (see FIG. 7). We asked first whetherboth EGF repeats 11 and 12 were necessary, and second, whether theflanking sequences from EGF repeats 10 and 13 were directly involved inbinding to Delta. Constructs #20 ΔCla+EGF(11-13), in which EGF repeat 12is the only entire repeat added, and #21 ΔCla+EGF(10-12), in which EGFrepeat 11 is the only entire repeat added, failed to mediateaggregation, suggesting that the presence of either EGF repeat 11 or 12alone was not sufficient for Notch-Delta interactions. However, sincethe 3' ligation juncture of these constructs interrupted the overallstructure of the EGF repeats, it was possible that a short "buffer" zonewas needed to allow the crucial repeat to function normally. Thus forexample in construct #19 ΔCla+EGF(10-13), EGF repeat 12 might not bedirectly involved in binding to Delta but instead might contribute theminimum amount of buffer sequence needed to protect the structure of EGFrepeat 11, thereby allowing interactions with Delta. Constructs #22-24addressed this issue. We designed PCR primers that broke at the end ofthe EGF repeat and therefore were less likely to disrupt the EGFdisulfide formation at the 3' ligation juncture. Constructs #22ΔCla+EGF(10-11), which did not mediate aggregation, and #23ΔCla+EGF(10-12), which did, again suggested that both repeats 11 and 12are required while the flanking sequence from repeat 13 clearly is not.Finally, construct #24 ΔCla+EGF(11-12), although now potentiallystructurally disrupted at the 5' junction, convincingly demonstratedthat the sequences from EGF repeat 10 are not crucial. Thus based onentirely consistent data from 24 constructs, we propose that EGF repeats11 and 12 of Notch together define the smallest functional unitobtainable from this analysis that contains the necessary sites forbinding to Delta in transfected S2 cells.

7.2.2. EGF REPEATS 11 AND 12 OF NOTCH ARE SUFFICIENT FOR NOTCH-DELTAMEDIATED AGGREGATION

The large ClaI deletion into which PCR fragments were inserted (#3 ΔCla)retains roughly 1/3 of the original 36 EGF repeats as well as the threeNotch/lin-12 repeats. While these are clearly not sufficient to promoteaggregation, it is possible that they form a necessary framework withinwhich specific EGF repeats can interact with Delta. To test whether onlya few EGF repeats were in fact sufficient to promote aggregation, wedesigned two constructs, #25 ΔEGF which deleted all 36 EGF repeatsexcept for the first two-thirds of repeat 1, and #30 ΔECN which deletedthe entire extracellular portion of Notch except for the first third ofEGF repeat 1 and ⁻ 35 amino acids just before the transmembrane domain.Fragments which had mediated Notch-Delta aggregation in the backgroundof construct #3 ΔCla, when inserted into construct #25 ΔEGF, were againable to promote interactions with Delta (constructs #26-30). Analogousconstructs (#31,32) in which the Notch/lin-12 repeats were also absent,again successfully mediated Notch-Delta aggregation. Thus EGF repeats 11and 12 appear to function as independent modular units which aresufficient to mediate Notch-Delta interactions in S2 cells, even in theabsence of most of the extracellular domain of Notch.

7.2.3. EGF REPEATS 11 AND 12 OF NOTCH MAINTAIN THE CALCIUM DEPENDENCE OFNOTCH-DELTA MEDIATED AGGREGATION

As described in Section 6, supra (Fehon et al., 1990, Cell 61, 523-534),we showed that Notch-Delta-mediated S2 cell aggregation is calciumdependent. We therefore examined the ability of cells expressing certaindeletion constructs to aggregate with Delta expressing cells in thepresence or absence of Ca⁺⁺ ions. We tested constructs #1 pMtNMg as acontrol, and #13, 16, 19, 23, 24, 26, 27 and 28, and found that cellsmixed in Ca⁺⁺ containing medium at 4° C. readily formed aggregates whilecells mixed in Ca⁺⁺ free medium containing EGTA failed to aggregate(Table III).

                  TABLE III    ______________________________________    EFFECT OF EXOGENOUS Ca.sup.++  ON    NOTCH - DELTA AGGREGATION.sup.a                Without Ca.sup.++  Ions                            With Ca.sup.++  Ions    ______________________________________     1. pMtNMg    0             37    13. ΔCla + EGF(9-17)                  0             31    16. ΔCla + EGF(9-13)                  0             38    19. ΔCla + EGF(10-13)                  0             42    23. ΔCla + EGF(10-12)                  0             48    29. ΔEGF + EGF(10-12)                  0             44    32. ΔECN + EGF(10-12)                  0             39    33. ΔCla + XEGF(10-13                  0             34    ______________________________________     .sup.a Data presented as percentage of Notchexpressing cells found in     aggregates (as in FIGS. 6A-6B).

Clearly, the calcium dependence of the interaction has been preserved ineven the smallest construct, consistent with the notion that the minimalconstructs containing EGF repeats 11 and 12 bind to Delta in a mannersimilar to that of full length Notch. This result is also interesting inlight of recent studies suggesting EGF-like repeats with a particularconsensus sequence may act as Ca⁺⁺ binding domains (Morita et al., 1984,J. Biol. Chem. 259, 5698-5704; Sugo et al., 1984, J. Biol. Chem. 259,5705-5710; Rees et al., 1988, EMBO J. 7, 2053-2061; Handford et al.,1990, EMBO J. 9, 475-480). Over half of the EGF repeats in Notch,including repeats 11 and 12, conform to this consensus, furtherstrengthening the argument that EGF repeats 11 and 12 are responsiblefor promoting Notch-Delta interactions.

7.2.4. THE DELTA BINDING FUNCTION OF EGF REPEATS 11 AND 12 OF NOTCH ISCONSERVED IN THE XENOPUS HOMOLOG OF NOTCH

Having mapped the Delta binding site to EGF repeats 11 and 12 of Notch,we were interested in asking whether this function was conserved in theNotch homolog that has been identified in Xenopus (Coffman et al., 1990,Science 249, 1438-1441). This protein shows a striking similarity toDrosophila Notch in overall structure and organization. For example,within the EGF repeat region both the number and linear organization ofthe repeats has been preserved, suggesting a possible functionalconservation as well. To test this, we made PCR primers based on theXenopus Notch sequence (Coffman et al., 1990, Science 249, 1438-1441)and used these to obtain an ˜350 bp fragment from a Xenopus Stage 17cDNA library that includes EGF repeats 11 and 12 flanked by half ofrepeats 10 and 13 on either side. This fragment was cloned intoconstruct #3 ΔCla, and three independent clones were tested for abilityto interact with Delta in the cell culture aggregation assay. Two of theclones, #33a&bΔCla+XEGF(10-13), when transfected into S2 cells were ableto mediate Notch-Delta interactions at a level roughly equivalent to theanalogous Drosophila Notch construct #l9ΔCla+EGF(10-13), and again in acalcium dependent manner (Table III). However, the third clone#33cΔCla+XEGF(10-13) failed to mediate Notch-Delta interactions althoughthe protein was expressed normally at the cell surface as judged bystaining live unpermeabilized cells. Sequence comparison of the XenopusPCR product in constructs #33a and 33c revealed a missense mutationresulting in a leucine to proline change (amino acid #453, Coffman, etal., 1990, Science 249, 1438-1441) in EGF repeat 11 of construct #33c.Although this residue is not conserved between Drosophila and XenopusNotch (FIG. 8), the introduction of a proline residue might easilydisrupt the structure of the EGF repeat, and thus prevent it frominteracting properly with Delta.

Comparison of the amino acid sequence of EGF repeats 11 and 12 ofDrosophila and Xenopus Notch reveals a high degree of amino acididentity, including the calcium binding consensus sequence (FIG. 8, SEQID NO:1 and NO:2). However the level of homology is not strikinglydifferent from that shared between most of the other EGF repeats, whichoverall exhibit about 50% identity at the amino acid level. This one toone correspondence between individual EGF repeats suggests that perhapsthey too may comprise conserved functional units.

7.3. DISCUSSION

We have continued our study of interactions between the protein productsof the genes Notch and Delta, using the in vitro S2 cell aggregationassay described in Section 6, supra. Based on an extensive deletionanalysis of the extracellular domain of Notch, we show that the regionsof Notch containing EGF-homologous repeats 11 and 12 are both necessaryand sufficient for Notch-Delta-mediated aggregation, and that this Deltabinding capability has been conserved in the same two EGF repeats ofXenopus Notch. Our finding that the aggregation mapped to EGF repeats 11and 12 of Notch demonstrates that the EGF repeats of Notch also functionas specific protein binding domains.

Recent studies have demonstrated that EGF domains containing a specificconsensus sequence can bind Ca⁺⁺ ions (Morita et al., 1984, J. Biol.Chem. 259, 5698-5704; Sugo et al., 1984, J. Biol. Chem. 259, 5705-5710;Rees et al., 1988, EMBO J. 7, 2053-2061; Handford et al., 1990, EMBO J.9, 475-480). In fact, about one half of the EGF repeats in Notch,including. repeats 11 and 12, conform to this consensus. We have shownthat exogenous Ca⁺⁺ was necessary for Notch-Delta mediated aggregationof transfected S2 cells (see Section 6; Fehon et al., 1990, Cell 61,523-534). We tested a subset of our deletion constructs and found thatEGF repeats 11 and 12 alone (#32ΔECN+EGF(11-12)) were sufficient tomaintain the Ca⁺⁺ dependence of Notch-Delta interactions.

A number of studies have suggested that the genetic interactions betweenNotch and Delta may reflect a dose sensitive interaction between theirprotein products. Genetic studies have indicated that the relative genedosages of Notch and Delta are crucial for normal development. Forexample, Xu et al. (1990, Genes Dev. 4, 464-475) found that nullmutations at Delta could suppress lethal interactions betweenheterozygous combinations of Abruptex (Ax) alleles, a class of Notchmutations that correlate with missense mutations within the EGF repeats(Hartley et al., 1987, EMBO J. 6, 3407-3417; Kelley et al., 1987, Mol.Cell Biol. 6, 3094-3108). The in vitro interactions we have described inwhich we observe both Notch-Delta and Delta--Delta associations (seeSection 6) imply that a competitive interaction between Notch and Deltafor binding to Delta may reflect the underlying basis for the observedgenetic interactions. Furthermore, we were able to coimmunoprecipitateNotch and Delta from both tissue culture and embryonic cell extracts(see Section 6), indicating a possible in vivo association of the twoproteins. In addition, mRNA in situ analyses of Notch and Deltaexpression patterns in the embryo suggest that expression of the two isoverlapping but not identical (Kopczynski and Muskavitch, 1989,Development 107, 623-636; Hartley et al., 1987, EMBO J. 6, 3407-3417).Detailed antibody analysis of Notch protein expression duringdevelopment have recently revealed Notch expression to be morerestricted at the tissue and subcellular levels than previous studieshad indicated (Johansen et al., 1989, J. Cell Biol. 109, 2427-2440; Kiddet al., 1989, Genes Dev. 3, 1113-1129).

Our finding that the same two EGF repeats from the Xenopus Notch homologare also able to mediate interactions with Delta in tissue culture cellsargues strongly that a similar function will have been conserved invivo. Although these two EGF repeats are sufficient in vitro, it is ofcourse possible that in vivo more of the Notch molecule may be necessaryto facilitate Notch-Delta interactions. In fact, we were somewhatsurprised for two reasons to find that the Delta binding site did notmap to EGF repeats where several of the Ax mutations have been shown tofall, first, because of the genetic screen (Xu et al., 1990, Genes Dev.4, 464-475) demonstrating interactions between Ax alleles and Deltamutations, and second, because of sequence analyses that have showncertain Ax alleles are associated with single amino acid changes withinthe putative Ca⁺⁺ binding consensus of the EGF repeats. For example, theAX^(E2) mutation changes EGF repeat 29 toward the Ca⁺⁺ binding consensussequence while the AX^(9B2) mutation moves EGF repeat 24 away from theconsensus. It is possible that in vivo these regions of the Notchprotein may be involved in interactions, either with Delta and/or otherproteins, that may not be accurately modelled by our cell culture assay.

Our in vitro mapping of the Delta binding domain to EGF repeats 11 and12 of Notch represents the first assignment of function to a structuraldomain of Notch. In fact, the various deletion constructs suggest thatthese two EGF repeats function as a modular unit, independent of theimmediate context into which they are placed. Thus, neither theremaining 34 EGF repeats nor the three Notch/lin-12 repeats appearnecessary to establish a structural framework required for EGF repeats11 and 12 to function. Interestingly, almost the opposite effect wasobserved: although our aggregation assay does not measure the strengthof the interaction, as we narrowed down the binding site to smaller andsmaller fragments, we observed an increase in the ability of thetransfected cells to aggregate with Delta expressing cells, suggestingthat the normal flanking EGF sequences actually impede associationbetween the proteins. For two separate series of constructs, either inthe background of construct #3 ΔCla (compare #9, 16, 19, 23) or in thebackground of construct #25 ΔEGF (compare #26, 27, 28), we observed anincrease in ability to aggregate such that the smallest constructs (#19,23, 28, 29) consistently aggregated above wild type levels (#1 pMtNMg).These results imply that the surrounding EGF repeats may serve to limitthe ability of EGF repeats 11 and 12 to access Delta, thereby perhapsmodulating Notch-Delta interactions in vivo.

Notch encodes a structurally complex transmembrane protein that has beenproposed to play a pleotropic role throughout Drosophila development.The fact that EGF repeats 11 and 12 appear to function as an independentmodular unit that is sufficient, at least in cell culture, forinteractions with Delta, immediately presents the question of the roleof the hypothesis is that these may also form modular binding domainsfor other proteins interacting with Notch at various times duringdevelopment.

In addition to Xenopus Notch, lin-12 and qlp-1, two genes thought tofunction in cell-cell interactions involved in the specification ofcertain cell fates during C. elegans development, encode EGF homologoustransmembrane proteins which are structurally quite similar toDrosophila and Xenopus Notch. All four proteins contain EGF homologousrepeats followed by three other cysteine rich repeats (Notch/lin-12repeats) in the extracellular domain, a single transmembrane domain, andsix cdc10/ankyrin repeats in the intracellular region. Unlike XenopusNotch, which, based on both sequence comparison as well as the resultsof our Delta binding assay, seems likely to encode the direct functionalcounterpart of Drosophila Notch, lin-12 and glp-1 probably encodedistinct members of the same gene family. Comparison of the predictedprotein products of lin-12 and glp-1 with Notch reveal specificdifferences despite an overall similar organization of structuralmotifs. The most obvious difference is that lin-12 and glp-1 proteinscontain only 13 and 10 EGF repeats, respectively, as compared to the 36for both Xenopus and Drosophila Notch. In addition, in the nematodegenes the array of EGF repeats is interrupted after the first EGF repeatby a distinct stretch of sequence absent from Notch. Furthermore, withrespect to the Delta binding domain we have defined as EGF repeats 11and 12 of Notch, there are no two contiguous EGF repeats in the lin-12or glp-1 proteins exhibiting the Ca⁺⁺ binding consensus sequence, norany two contiguous repeats exhibiting striking similarity to EGF repeats11 and 12 of Notch, again suggesting that the lin-12 and glp-1 geneproducts are probably functionally distinct from Notch.

Our finding that EGF repeats 11 and 12 of Notch form a discrete Deltabinding unit represents the first concrete evidence supporting the ideathat each EGF repeat or small subset of repeats may play a unique roleduring development, possibly through direct interactions with otherproteins. The homologies seen between the adhesive domain of Delta andSerrate (see Section 8.3.4, infra) suggest that the homologous portionof Serrate is "adhesive" in that it mediates binding to othertoporythmic proteins. In addition, the gene scabrous, which encodes asecreted protein with similarity to fibrinogen, may interact with Notch.

In addition to the EGF repeat, multiple copies of other structuralmotifs commonly occur in a variety of proteins. One relevant example isthe cdc10/ankyrin motif, six copies of which are found in theintracellular domain of Notch. Ankyrin contains 22 of these repeats.Perhaps repeated arrays of structural motifs may in general represent alinear assembly of a series of modular protein binding units. Giventhese results together with the known structural, genetic anddevelopmental complexity of Notch, Notch may interact with a number ofdifferent ligands in a precisely regulated temporal and spacial patternthroughout development. Such context specific interactions withextracellular proteins could be mediated by the EGF and Notch/lin-12repeats, while interactions with cytoskeletal and cytoplasmic proteinscould be mediated by the intracellular cdc10/ankyrin motifs.

8. THE AMINO-TERMINUS OF DELTA IS AN EGF-BINDING DOMAIN THAT INTERACTSWITH NOTCH AND DELTA

Aggregation of cultured cells programmed to express wild type andvariant Delta proteins has been employed to delineate Delta sequencesrequired for heterotypic interaction with Notch and homotypic Deltainteraction. We have found that the amino terminus of the Deltaextracellular domain is necessary and sufficient for the participationof Delta in heterotypic (Delta-Notch) and homotypic (Delta--Delta)interactions. We infer that the amino terminus of Delta is an EGFmotif-binding domain (EBD), given that Notch EGF-like sequences aresufficient to mediate heterotypic interaction with Delta. The Delta EBDapparently possesses two activities: the ability to bind EGF-relatedsequences and the ability to self-associate. We also find that Delta istaken up by cultured cells that express Notch, which may be a reflectionof a mechanism by which these proteins interact in vivo.

8.1. MATERIALS AND METHODS

8.1.1. CELL LINES

The S2 Drosophila cell line (Schneider, 1972, J. Embryol. Exp. Morph.27, 353-365)) used in these experiments was grown as described inSection 6.

8.1.2. IMMUNOLOGICAL PROBES

Immunohistochemistry was performed as described in Section 6, supra, orsometimes with minor modifications of this procedure. Antisera andantibodies employed included mouse polyclonal anti-Delta sera raisedagainst a Delta ELR array segment that extends from the fourth throughninth ELRs (see Section 6); rat polyclonal anti-Delta sera raisedagainst the same Delta segment (see Section 6); rat polyclonalanti-Notch sera raised against a Notch ELR array segment that extendsfrom the fifth through thirteenth ELRs; mouse monoclonal antibodyC17.9C6 (see Section 6), which recognizes the Notch intracellulardomain; and mouse monoclonal antibody BP-104 (Hortsch et al., 1990,Neuron 4, 697-709), which recognizes the long form of Drosophilaneuroglian.

8.1.3. EXPRESSION VECTOR CONSTRUCTS

Constructs employed to program expression of wild type Delta (pMTDl1)and wild type Notch (pMTNMg) are described in Section 6, supra.Constructs that direct expression of variant Delta proteins weregenerated using pMTDl1, the Dl1 cDNA cloned into Bhluescript+ (pBSDll;Kopczynski et al., 1988, Genes Dev. 2, 1723-1735), and pRmHa3-104 (A. J.Bieber, pers. comm.), which consists of the insertion of the 1B7A-250cDNA into the metallothionein promoter vector pRmHa-3 (Bunch et al.,1988, Nucl. Acids Res. 16, 1043-1061) and supports inducible expressionof the long form of Drosophila neuroglian (Hortsch et al., 1990, Neuron4, 697-709).

Briefly, constructs were made as follows:

Del(Sca-Nae)--Cut pBSDl1 with SalI (complete digest) and ScaI (partial),isolate vector-containing fragment. Cut pBSDl1 with NaeI (partial) andSalI (complete), isolate Delta carboxyl-terminal coding fragment. Ligatefragments, transform, and isolate clones. Transfer EcoRI insert intopRmHa-3.

Del(Bam-Bgl)--Cut pBSDl1 with BglII (complete) and BamHI (partial), fillends with Klenow DNA polymerase, ligate, transform, and isolate clones.Transfer EcoRI insert into pRmHa-3.

Del(ELR1-ELR3)--PCR-amplify basepairs 236-830 of the Dl1 CDNA using5-ACTTCAGCAACGATCACGGG-3' (SEQ ID NO:26) and 5'-TTGGGTATGTGACAGTAATCG-3'(SEQ ID NO:27), treat with T4 DNA polymerase, ligate into pBSDl1 cutwith ScaI (partial) and BglII (complete) and end-filled with Klenow DNApolymerase, transform, and isolate clones. Transfer BamHI-SalI Deltacarboxyl-terminal coding fragment into pRmHa-3.

Del(ELR4-ELR5)--pBSDl1 was digested to completion with BglII andpartially with PstI. The 5.6 kb vector-containing fragment was isolated,circularized using T4 DNA ligase in the presence of a 100× molar excessof the oligonucleotide 5'-GATCTGCA-3', and transformed and clones wereisolated. The resulting EcoRI insert was then transferred into pRmHa-3.

Ter(Dde)--Cut pBSDl1 with DdeI (partial), end-fill with Klenow DNApolymerase, ligate with 100× molar excess of 5'-TTAAGTTAACTTAA-3' (SEQID NO:28), transform, and isolate clones. Transfer EcoRI insert intopRmHa-3.

Ins(Nae)A--Cut pMTDl1 with NaeI (partial), isolate vector-containingfragment, ligate with 100× molar excess of 5'-GGAAGATCTTCC-3' (SEQ IDNO:29), transform, and isolate clones.

NAE B--pMTDl1 was digested partially with NaeI, and the population oftentatively linearized circles approximately 5.8 kb in length wasisolated. The fragments were recircularized using T4 DNA ligase in thepresence of a 100× molar excess of the oligonucleotide5'-GGAAGATCTTCC-3' (SEQ ID NO:29) and transformed, and a clone (NAE A)that contained multiple inserts of the linker was isolated. NAE A wasdigested to completion with BglII, and the resulting 0.4 kb and 5.4 kbfragments were isolated, ligated and transformed, and clones wereisolated.

Ins(Stu)--Cut pMTDl1 with StuI (complete), isolate vector-containingfragment, ligate with 100× molar excess of 5'-GGAAGATCTTCC-3'(SEQ IDNO:29), transform and isolate clones.

STU B--pMTDl1 was digested completely with StuI, and the resulting 5.8kb fragment was isolated. The fragment was recircularized using T4 DNAligase in the presence of a 100× molar excess of the oligonucleotide5'-GGAAGATCTTCC-3' (SEQ ID NO:29) and transformed, and a clone (STU A)that contained multiple inserts of the linker was isolated. STU B wasdigested to completion with BglII, and the resulting 0.6 kb and 5.2 kbfragments were isolated, ligated and transformed, and clones wereisolated.

NG1--Cut pRmHa3-104 with BglII (complete) and EcoRI (complete), isolatevector-containing fragment. Cut Ins(Nae)A with EcoRI (complete) andBglII (complete), isolate Delta amino-terminal coding fragment. Ligatefragments, transform and isolate clones.

NG2--Cut pRmHa3-104 with BglII (complete) and EcoRI (complete), isolatevector-containing fragment. Cut Del(ELR1-ELR3) with EcoRI (complete) andBglII (complete), isolate Delta amino-terminal coding fragment. Ligatefragments, transform and isolate clones.

NG3--Cut pRmHa3-104 with BglII (complete) and EcoRI (complete), isolatevector-containing fragment. Cut pMTDl1 with EcoRI (complete) and BglII(complete), isolate Delta amino-terminal coding fragment. Ligatefragments, transform and isolate clones.

NG4--Cut pRmHa3-104 with BglII (complete) and EcoRI (complete), isolatevector containing fragment. Cut Del(Sca-Nae) with EcoRI (complete) andBglII (complete), isolate Delta amino-terminal coding fragment. Ligatefragments, transform and isolate clones.

NG5--Generate Del(Sca-Stu) as follows: cut pMTDl1 with ScaI (complete)and StuI (complete), isolate ScaI-ScaI amino-terminal coding fragmentand StuI-ScaI carboxyl-terminal coding fragment, ligate, transform andisolate clones. Cut Del(Sca-Stu) with EcoRI (complete) and BglII(complete), isolate Delta amino terminal coding fragment. Cut pRmHa3-104with BglII (complete) and EcoRI (complete), isolate vector-containingfragment. Ligate fragments, transform and isolate clones.

The sequence contents of the various Delta variants are shown in TableIV. Schematic diagrams of the Delta variants defined in Table IV areshown in FIG. 9C.

                  TABLE IV    ______________________________________    SEQUENCE CONTENTS OF DELTA VARIANTS    EMPLOYED IN THIS STUDY             Nucleotides     Amino Acids    ______________________________________    Wild type  1-2892.sup.A      1-833    Del(Sca-Nae)               1-235/734-2892    1-31/W/199-                                 833    Del(Bam-Bgl)               1-713/1134-2892   1-191/332-833    Del(ELR1-ELR3)               1-830/1134-2892   1-230/332-833    Del(ELR4-ELR5)               1-1137/1405-2892  1-332/422-833    Ter(Dde)   1-2021/TTAAGTTAACTTAA.sup.E /                                 1-626/H               2227-2892    Ins(Nae)A  1-733/(GGAAGATCTTCC).sub.n .sup.F /                                 1-197/(RKIF).sub.n               734-2892.sup.B    198-833    NAE B      1-733/GGAAGATCTTCC.sup.F /                                 1-197/RKIF               734-2892          198-833    Ins(Stu)   1-535/(GGAAGATCTTCC).sub.n .sup.F /                                 1-131/               536-2892.sup.B    G(KIFR).sub.n-1                                 KIFP/133-833    STU B      1-535/GGAAGATCTTCC.sup.F /                                 1-131/GKIFP               536-2892          133-833    NG1        1-733/GGAA/2889-3955(NG).sup.c                                 1-198/K/ 952-                                 1302.sup.D    NG2        1-830/ 2889-3955(NG)                                 1-230/952-                                 1302    NG3        1-1133/2889-3955(NG)                                 1-331/952-                                 1302    NG4        1-235/734-1133/   1-31/199-331/               2889-3955(NG)     952-1302    NG5        1-235/536-1133/   1-31/S/133-               2889-3955(NG)     952-1302    ______________________________________     .sup.A Coordinates for Delta sequences correspond to the sequence of the     Dl1 cDNA (FIGS. 12A-12C).     .sup.B The exact number of linkers inserted has not been determined for     this construct.     .sup.C Coordinates for neuroglian (Bieber et al., 1989, Cell 59, 447-460;     Hortsch et al., 1990, Neuron 4, 697-709) nucleotide sequences present in     Deltaneuroglian chimeras correspond to the sequence of the 1B7A250 cDNA     (FIGS. 13A-13F, SEQ ID NO:5) and are indicated in bold face type.     .sup.D Neuroglian amino acid sequences are derived from conceptual     translation of the 1B7A250 cDNA nucleotide sequence (FIGS. 13A-13F, SEQ I     NO:5) and are indicated in bold face type.     .sup.E SEQ ID NO:28     .sup.F SEQ ID NO:29

8.1.4. AGGREGATION PROTOCOLS

Cell transfection and aggregation were performed as described in Section6, supra, or with minor modifications thereof.

8.2. RESULTS

8.2.1. AMINO-TERMINAL SEQUENCES WITHIN THE DELTA EXTRACELLULAR DOMAINARE NECESSARY AND SUFFICIENT FOR THE HETEROTYPIC INTERACTION WITH NOTCH

Because we anticipated that some Delta variants might not be efficientlylocalized on the cell surface, we investigated the relationship betweenthe level of expression of wild type Delta and the extent of aggregationwith Notch-expressing cells by varying the input amount of Deltaexpression construct in different transfections. We found that theheterotypic Delta-Notch interaction exhibits only slight dependence onthe Delta input level over a 10-fold range in this assay (FIGS. 9A-9B).Given the robustness of the heterotypic interaction over the rangetested and our observations that each of the Delta variants we employedexhibited substantial surface accumulation in transfected cells, weinfer that the inability of a given Delta variant to support heterotypicaggregation most probably reflects a functional deficit exhibited bythat variant, as opposed to the impact of reduced levels of surfaceexpression on heterotypic aggregation.

The results of the heterotypic aggregation experiments mediated by Deltavariants and wild-type Notch are shown in Table V.

                                      TABLE V    __________________________________________________________________________    HETEROTYPIC AGGREGATION MEDIATED BY    DELTA VARIANTS AND WILD TYPE NOTCH             Aggregated                   Unaggregated                              Aggregated                                      Unaggregated    Construct             Total.sup.A                   Total  Expt. #                              Notch.sup.+                                  Delta.sup.+                                      Notch.sup.+B                                          Delta.sup.+    __________________________________________________________________________    Wild type             33 (H).sup.C                   179    1   15  18   67 112             58 (H)                   247    2   37  21  218 29             38 (H)                   209    3   21  17  148 61             29 (H)                   174    4   18  11   95 79             175                (B)                    68    5   84  91   37 31    Del (Sca--Nae)             0  (H)                   207    1    0   0  125 82             0  (H)                   226    2    0   0  215 11             0  (H)                   287    3    0   0  215 72             0  (H)                   200    4    ND.sup.D                                  ND  ND  ND    Del (Bam--Bgl)             4  (H)                   245    1    3   1  171 74             0  (H)                   200    2    0   0  110 90             0  (H)                   200    3   ND  ND  ND  ND    Del (ELR1-ELR3)             28 (B)                   296    1   11  17  139 157             20 (B)                    90    2    9  11   53 37             22 (B)                   227    3   19  13  114 113             127                (B)                    97    4   19  78   66 61    Del (ELR4-ELR5)             38 (H)                   188    1   26  12  141 47             36 (H)                   204    2   20  16   90 114    Ter (Dde)             53 (H)                   236    1   24  29  144 92             51 (H)                   214    2   30  21  126 88             52 (H)                   190    3   30  22  110 80    Ins (Nae) A             0  (B)                   205    1    0   0  111 94             0  (B)                   254    2    0   0  161 93             0  (B)                   201    3    0   0  121 80    NG1      0  (B)                   208    1    0   0  140 68             0  (B)                   114    2    0   0   38 76             0  (B)                   218    3    0   0   76 142    NG2      14 (B)                   106    1    7   7   54 52             50 (B)                   216    2   35  15   94 122             36 (B)                   168    3   12  24   29 139    NG3      71 (B)                   175    1   43  28   84 91    NG4      0  (B)                   254    1    0   0  150 104             0  (B)                   215    2    0   0   35 180             0  (B)                   200    3    0   0   93 107    __________________________________________________________________________     .sup.A Total number of expressing cells in aggregates that contain four o     more cells.     .sup.B Cells that express neuroglianbased constructs (NGn) were detected     using a monoclonal antibody that recognizes the intracellular domain of     neuroglian (see Materials and Methods).     .sup.C (H) indicates that cells were aggregated in a 25 ml Erlenmeyer     flask, (B) indicates that cells were aggregated in a 12well microtiter     plate (see Materials and Methods).     .sup.D Data for individual cell types (i.e., Delta.sup.+  and Notch.sup.+     in aggregates and unaggregated were not recorded.

Delta amino acids (AA) 1-230 is the current minimum sequence intervaldefined as being sufficient for interaction with Notch. This is based onthe success of NG2-Notch aggregation. Within this interval, DeltaAA198-230 are critical because their deletion in the NG1 constructinactivated the Notch-binding activity observed for the NG2 construct.Also within this interval, Delta AA32-198 are critical because theirdeletion in the NG4 construct also inactivated the Notch-bindingactivity observed for the NG3 construct. The importance of DeltaAA192-230 is also supported by the observation that the Del(ELR1-ELR3)variant, which contains all Delta amino acids except AA231-331,possessed Notch-binding activity, while the Del(Bam-Bgl) variant, whichcontains all Delta amino acids except AA192-331, was apparentlyinactivated for Notch-binding activity.

Conformation and/or primary sequence in the vicinity of Delta AA197/198is apparently critical because a multimeric insertion of thetetrapeptide -Arg-Lys-Ile-Phe in one letter code (see e.g. Lehninger etal., 1975, Biochemistry, 2d ed., p. 72), RKIF! (SEQ ID NO:30)--betweenthese two residues, as in the Ins(Nae)A construct, inactivated theNotch-binding activity observed with wild type Delta.

In addition, the observation that the Del(ELR1-ELR3) construct supportedaggregation implies that ELR1-ELR3 are not required for Delta-Notchinteraction; the observation that the Del(ELR4-ELR5) construct supportedaggregation implies that ELR4 and ELR5 are not required for Delta-Notchinteraction, and the observation that the Ter(Dde) construct supportedaggregation implies that the Delta intracellular domain is not requiredfor Delta-Notch interaction.

8.2.2. AMINO-TERMINAL SEQUENCES WITHIN THE DELTA EXTRACELLULAR DOMAINARE NECESSARY AND SUFFICIENT FOR HOMOTYPIC INTERACTION

The results of the homotypic aggregation experiments mediated by Deltavariants is shown in Table VI.

                  TABLE VI    ______________________________________    HOMOTYPIC AGGREGATION MEDIATED BY DELTA VARIANTS    Construct   Aggregated  Unaggregated                                       Expt. #    ______________________________________    Wild type   38      (H).sup.A                                175        1                48      (H)     171        2                13      (H)     95         3                33      (H)     173        4                134     (B)     72         5    Del(Sca-Nae)                0       (H)     200        1                0       (H)     200        2                0       (H)     200        3    Del(Bam-Bgl)                0       (H)     200        1                0       (H)     200        2                0       (H)     200        3    Del(ELR1-ELR3)                160     (B)     62         1                55      (B)     80         2                0       (B)     200        3                4       (B)     203        4                41      (B)     234        5                4       (B)     366         6.sup.B                23      (B)     325  (1:20)                0       (B)     400         7.sup.B                5       (B)     347  (1:5)                10      (B)     228  (1:20)                0       (B)     400         8.sup.B                16      (B)     346  (1:5)                4       (B)     268  (1:20)                4       (B)     500         9.sup.C                18      (B)     500  (1:5)                12      (B)     271  (1:20)                7       (B)     128  (1:50)                0       (B)     500        10.sup.C                0       (B)     500  (1:5)                0       (B)     500  (1:20)                21      (B)     246  (1:50)                0       (B)     500        11.sup.C                5       (B)     500  (1:5)                8       (B)     177  (1:20)                4       (B)     69   (1:50)    Del(ELR4-ELR5)                21      (H)     175        1                29      (H)     243        2                35      (H)     179        3    Ter(Dde)    53      (H)     164        1                33      (H)     178        2                36      (H)     203        3    Ins(Nae)A   0       (B)     200        1                0       (B)     200        2                0       (B)     200        3    ______________________________________     .sup.A (H) indicates that cells were aggregated in a 25 ml Erlenmeyer     flask; (B) indicates that cells were aggregated in a 12well microtiter     plate (see Materials and Methods).     .sup.B Transfected cells were incubated under aggregation conditions     overnight, then diluted into the appropriate volume of logphase S2 cells     in the presence of inducer and incubated under aggregation conditions for     an additional four to six hours.     .sup.C Transfected cells to which inducer had been added were diluted int     the appropriate volume of logphase S2 cells to which inducer had been     added, and the cell mixture was incubated under aggregation conditions     overnight.

Deletion of Delta AA32-198 Del(Sca-Nae)! or Delta AA192-331Del(Bam-Bgl)! from the full-length Delta protein eliminated theDelta--Delta interaction. Deletion of Delta AA231-331 Del(ELR1-ELR3)!did not eliminate the Delta--Delta interaction. Therefore, sequenceswithin the Delta AA32-230 are required for the Delta--Delta interaction.

Conformation and/or primary sequence in the vicinity of Delta AA197/198is apparently critical for the Delta--Delta interaction because amultimeric insertion of the tetrapeptide -Arg-Lys-Ile-Phe- (SEQ IDNO:30) between these two residues, as in the Ins(Nae)A construct,inactivated Delta--Delta interaction.

In addition, the observation that the Del(ELR1-ELR3) construct couldsupport aggregation implies that ELR1-ELR3 are not required forDelta--Delta interaction; the observation that the Del(ELR-ELR5)construct supported aggregation implies that ELR4 and ELR5 are notrequired for Delta--Delta interaction, and the observation that theTer(Dde) construct supported aggregation implies that the Deltaintracellular domain is not required for Delta--Delta interaction.

A summary of the results of assays for heterotypic and homotypicaggregation with various constructs are shown in Table VI A.

                  TABLE VI A    ______________________________________    AGGREGATION MEDIATED BY WILD    TYPE AND VARIANT DELTA PROTEINS             HETEROTYPIC   HOMOTYPIC             AGGREGATION.sup.a                           AGGREGATION.sup.b    CONSTRUCT  DELTA     NOTCH     DELTA    ______________________________________    Wild Type   33 ± 12.sup.c                          26 ± 11.sup.c                                    27 ± 10.sup.c    Del(Sca-Nae)               0         0         0    Del(Bam-Bgl)                0.4 ± 0.4                          0.6 ± 0.6                                   0    Del(ELR1-ELR3)                25 ± 11.sup.d                          15 ± 3.sup.d                                    32 + 15.sup.d    Del(ELR4-ELR5)               17 ± 2 18 ± 2 13 ± 2    Ter(Dde)   22 ± 1 18 ± 2 18 ± 3    NAE B      25 ± 5 0         27 ± 7    STU B      0         0         0    NG1        0         0         0    NG2        13 ± 1 23 ± 6  4 ± 1.sup.d    NG3        16 ± 1 13 ± 1 27 ± 17    NG4        0         0         0.5 ± 0.3    ______________________________________     .sup.a Mean fraction (%) of Delta or Notch cells in aggregates of four or     more cells (± standard error). N = 3 replicates, unless otherwise     noted.     .sup.b Mean fraction (%) of Delta cells in aggregates of four or more     cells (± standard error). N = 3 replicates, unless otherwise noted.     .sup.c N = 5 replicates.     .sup.d N = 4 replicates.

8.2.3. DELTA SEQUENCES INVOLVED IN HETEROTYPIC AND HOMOTYPICINTERACTIONS ARE QUALITATIVELY DISTINCT

The respective characteristics of Delta sequences repaired forheterotypic and homotypic interaction were further defined using Deltavariants in which short, in-frame, translatable linker insertions wereintroduced into the Delta amino terminus (i.e., NAE B and STU B; FIG.9C, Table VI A). Replacement of Delta residue 132 (A) with thepentapeptide GKIFP (STU B variant) leads to the inactivation ofheterotypic and homotypic interaction activities of the Delta aminoterminus. This suggests that some Delta sequences required for these twodistinct interactions are coincident and reside in proximity to residue132. On the other hand, insertion of the tetrapeptide RKIF between Deltaresidues 198 and 199 (NAE B variant) eliminates the ability of the Deltaamino terminus to mediate heterotypic interaction with Notch, but has noapparent effect on the ability of the altered amino terminus to mediatehomotypic interaction. The finding that the NAE B insertion affects onlyone of the two activities of the Delta amino terminus implies that theDelta sequences that mediate heterotypic and homotypic interactions,while coincident, are qualitatively distinct.

8.2.4. DELTA IS TAKEN UP BY CELLS THAT EXPRESS NOTCH

During the course of many heterotypic aggregation experiments, we havenoted that Delta protein can sometimes be found within cells that havebeen programmed to express Notch, but not Delta. We conduct heterotypicaggregation assays by mixing initially separate populations of S2 cellsthat have been independently transfected with expression constructs thatprogram expression of either Delta or Notch. Yet, we often detectpunctate staining of Delta within Notch-expressing cells found inheterotypic aggregates using Delta-specific antisera. Our observationsare consistent with Delta binding directly to Notch at the cell surfaceand subsequent clearance of this Delta-Notch complex from the cellsurface via endocytosis.

8.3. DISCUSSION

8.3.1. AMINO-TERMINAL SEQUENCES UNRELATED TO EGF ARE INVOLVED IN THEINTERACTION BETWEEN DELTA AND NOTCH

We have employed cell aggregation assays to define a region within theamino-proximal region of the Delta extracellular domain that isnecessary and sufficient to mediate the Delta-Notch interaction.Functional analyses of a combination of deletion and sufficiencyconstructs revealed that this region extends, maximally, from AAlthrough AA230. It is striking that this region does not include any ofthe EGF-like sequences that reside within the Delta extracellulardomain. It is probable that the particular Delta sequences within thesufficient interval required for interaction with Notch includeAA198-230 because deletion of these residues eliminates Notch-bindingactivity. The fact that deletion of AA32-198 also inactivatesNotch-binding activity suggests that sequences amino-proximal to AA198are also required, although the deleterious impact of this deletioncould result from the removal of additional amino acids in the immediatevicinity of AA198.

Sequences within Delta sufficient for interaction with Notch can begrouped into three subdomains--N1, N2, and N3--that differ in theirrespective contents of cysteine residues (FIG. 10, SEQ ID NO:3). The N1and N3 domains each contain six cysteine residues, while the N2 domaincontains none. The even number of cysteines present in N1 and N3,respectively, allows for the possibility that the respective structuresof these subdomains are dictated, in part, by the formation ofparticular disulfide bonds. The broad organizational pattern of theDelta amino-terminus is also generally analogous to that of theextracellular domain of the vertebrate EGF receptor (Lax et al., 1988,Mol. Cell. Biol. 8, 1970-1978), in which sequences believed to interactwith EGF are bounded by two cysteine-rich subdomains.

8.3.2. DELTA SEQUENCES REQUIRED FOR HOMOTYPIC AND FOR HOMOTYPICHETEROTYPIC INTERACTIONS APPEAR TO BE COINCIDENT

Our results also indicate that sequences essential for homotypic Deltainteraction reside within the interval AA32-230. Deletion of sequencesor insertion of additional amino acids within this amino-proximal domaineliminate the ability of such Delta variants to singly promote cellaggregation. Thus, sequences required for Dleta--Delta interaction mapwithin the same domain of the protein as those required for Delta-Notchinteraction.

8.3.3. THE DELTA AMINO TERMINUS CONSTITUTES AN EGF-BINDING MOTIF

The work described in examples supra has revealed that Notch sequencesrequired for Delta-Notch interaction in the cell aggregation assay mapwithin the EGF-like repeat array of the Notch extracellular domain. Thisfinding implies that Delta and Notch interact by virtue of the bindingof the Delta amino-terminus to EGF-like sequences within Notch and,therefore, that the amino-terminus of the Delta extracellular domainconstitutes an EGF-binding domain (FIGS. 11A-11B).

These results also raise the possibility that homotypic Deltainteraction involves the binding of the Delta amino-terminus to EGF-likesequences within the Delta extracellular domain (FIGS. 12A-12C).However, none of the EGF-like repeats within the Delta extracellulardomain are identical to any of the EGF-like repeats within the Notchextracellular domain (FIGS. 13A-13F, SEQ ID NO:6; Wharton et al., 1985,Cell 43, 567-581). Given this fact, if Delta homotypic interactions areindeed mediated by interaction between the Delta amino-terminus andDelta EGF-like repeats, then the Delta EGF-binding domain has thecapacity to interact with at least two distinct EGF-like sequences.

8.3.4. DELTA SEQUENCES INVOLVED IN THE DELTA-NOTCH INTERACTION ARECONSERVED IN THE SERRATE PROTEIN

Alignment of amino acid sequences from the amino termini of the Delta(FIGS. 13A-13F, SEQ ID NO:6, and FIGS. 15A-15B, SEQ ID NO:9) and Serrate(Fleming et al., 1990, Genes & Dev. 4, 2188-2201; Thomas et al., 1991,Devel. 111, 749-761) reveals a striking conservation of structuralcharacter and sequence composition. The general N1-N2-N3 subdomainstructure of the Delta amino terminus is also observed within theSerrate amino terminus, as is the specific occurrence of six cysteineresidues within the Delta N1- and Delta N3-homologous domains of theSerrate protein. Two notable blocks of conservation correspond to DeltaAA63-73 (8/11 residues identical) and Delta AA195-206 (10/11 residuesidentical). The latter block is of particular interest because insertionof additional amino acids in this interval can eliminate the ability ofDelta to bind to Notch or Delta.

8.3.5. CIS AND TRANS INTERACTIONS BETWEEN DELTA AND NOTCH MAY INVOLVEDIFFERENT SEQUENCES WITHIN NOTCH

Inspection of the overall structures of Delta and Notch suggests thatDelta-Notch interaction could involve contacts between the DeltaEGF-binding domain with either of two regions within Notch, depending onwhether the interaction were between molecules that reside on opposingmembranes or within the same membrane (FIGS. 11A-11B). The cellaggregation assays, which presumably detect the interaction of moleculesin opposing membranes, imply that the Delta EGF-binding domain interactswith Notch EGF-like repeats 11 and 12 (see examples supra). If tandemarrays of EGF-like motifs do form rod-like structures (Engel, 1989, FEBSLett. 251, 1-7) within the Delta and Notch proteins, then the estimateddisplacement of the Delta EGF-binding domain from the cell surface wouldpresumably be sufficient to accommodate the rigid array of NotchEGF-like repeats 1-10. It is also intriguing to note that thedisplacement of the Delta EGF-binding domain from the cell surface couldplace this domain in the vicinity of the Notch EGF-like repeats (25-29)that are affected by Abruptex mutations (Hartley et al., 1987, EMBO J.6, 3407-3417; Kelley et al., 1987, Mol. Cell. Biol. 6, 3094-3108) andcould allow for interaction of Delta and Notch proteins present withinthe same membrane.

8.3.6. INTERACTIONS ANALOGOUS TO THE DELTA-NOTCH INTERACTION INVERTEBRATES

Given the interaction between Delta and Notch in Drosophila, it is quiteprobable that a Delta homologue (Helta?) exists in vertebrates and thatthe qualitative and molecular aspects of the Delta-Notch andDelta--Delta interactions that we have defined in Drosophila will behighly conserved in vertebrates, including humans. Such homologs can becloned and sequenced as described supra, Section 5.2.

9. SEQUENCES WHICH MEDIATE NOTCH-SERRATE INTERACTIONS

We report a novel molecular interaction between Notch and Serrate, andshow that the two EGF repeats of Notch which mediate interactions withDelta, namely EGF repeats 11 and 12, also constitute a Serrate bindingdomain.

To test whether Notch and Serrate directly interact, S2 cells weretransfected with a Serrate expression construct and mixed with Notchexpressing cells in our aggregation assay. For the Serrate expressionconstruct, a synthetic primer containing an artificial BamHI siteimmediately 5' to the initiator AUG at position 442 (all sequencenumbers are according to Fleming et al., 1990, Genes & Dev. 4:2188-2201)and homologous through position 464, was used in conjunction with asecond primer from position 681-698 to generate a DNA fragment of ˜260base pairs. This fragment was cut with BamHI and KpnI (position 571) andligated into Bluescript KS+ (Stratagene). This construct, BTSer5'PCR,was checked by sequencing, then cut with KpnI. The Serrate KpnI fragment(571-2981) was inserted and the proper orientation selected, to generateBTSer5'PCR-Kpn. The 5' SacII fragment of BTSer5'PCR-Kpn (SacII sites inBluescript polylinker and in Serrate (1199)) was isolated and used toreplace the 5' SacII fragment of cDNA C1 (Fleming et al., 1990, Genes &Dev. 4:2188-2201), thus regenerating the full length Serrate cDNA minusthe 5' untranslated regions. This insert was isolated by a SalI andpartial BamHI digestion and shuttled into the BamHI and SalI sites ofpRmHa-3 to generate the final expression construct, Ser-mtn.

We found that Serrate expressing cells adhere to Notch expressing cellsin a calcium dependent manner (FIGS. 6A-6B and Table VII). However,unlike Delta, under the experimental conditions tested, Serrate does notappear to interact homotypically. In addition, we detect no interactionsbetween Serrate and Delta.

                  TABLE VII    ______________________________________    Effect of Exogenous Ca.sup.++  on Notch - Serrate    Aggregation.sup.a                   Notch-Serrate                   Without Ca.sup.++                            With Ca.sup.++    ______________________________________     1. pMtNMg       0          15    32. ΔECN + EGF(10-12)                     0          13    33. ΔCla + XEGF(10-13)                     0          15    ______________________________________     .sup.a Data presented as percentage of Notch expressing cells found in     aggregates (as in FIGS. 6A-6B). All numbers are from single transfection     experiments (rather than an average of values from several separate     experiments as in FIGS. 6A-6B).

We have tested a subset of our Notch deletion constructs to map theSerrate-binding domain and have found that EGF repeats 11 and 12, inaddition to binding to Delta, also mediate interactions with Serrate(FIGS. 6A-6B; Constructs #1, 7-10, 13, 16, 17, 19, 28, and 32). Inaddition, the Serrate-binding function of these repeats also appears tohave been conserved in the corresponding two EGF repeats of XenopusNotch (#33ΔCla+XEGF(10-13)). These results unambiguously show that Notchinteracts with both Delta and Serrate, and that the same two EGF repeatsof Notch mediate both interactions. We were also able to define theSerrate region which is essential for the Notch/Serrate aggregation.Deleting nucleotides 676-1287 (i.e. amino acids 79-282) (See FIGS.15A-15B) eliminates the ability of the Serrate protein to aggregate withNotch.

Notch and Serrate appear to aggregate less efficiently than Notch andDelta, perhaps because the Notch-Serrate interaction is weaker. Forexample, when scoring Notch-Delta aggregates, we detect ˜40% of allNotch expressing cells in clusters with Delta expressing cells (FIGS.6A-6B, #1 pMtNMg) and ˜40% of all Delta expressing cells in contact withNotch expressing cells. For Notch-Serrate, we find only ˜20% of allNotch expressing cells (FIGS. 6A-6B; pMtNMg) and ˜15% of all Serrateexpressing cells in aggregates. For the various Notch deletionconstructs tested, we consistently detect a reduction in the amount ofaggregation between Notch and Serrate as compared to the correspondingNotch-Delta levels (FIGS. 6A-6B), with the possible exception ofconstructs #9 and 10 which exhibit severely reduced levels ofaggregation even with Delta. One trivial explanation for this reducedamount of aggregation could be that our Serrate construct simply doesnot express as much protein at the cell surface as the Delta construct,thereby diminishing the strength of the interaction. Alternatively, thedifference in strength of interaction may indicate a fundamentalfunctional difference between Notch-Delta and Notch-Serrate interactionsthat may be significant in vivo.

10. THE CLONING, SEQUENCING, AND EXPRESSION OF HUMAN NOTCH

10.1. ISOLATION AND SEQUENCING OF HUMAN NOTCH

Clones for the human Notch sequence were originally obtained using thepolymerase chain reaction (PCR) to amplify DNA from a 17-18 week humanfetal brain cDNA library in the Lambda Zap II vector (Stratagene).Degenerate primers to be used in this reaction were designed bycomparing the amino acid sequences of the Xenopus homolog of Notch withDrosophila Notch. Three primers (cdc1 (SEQ ID NO:10), cdc2 (SEQ IDNO:11), and cdc3 (SEQ ID NO:12); FIGS. 16A-16C) were designed to amplifyeither a 200 bp or a 400 bp fragment as primer pairs cdc1/cdc2 orcdc1/cdc3, respectively.

The 400 bp fragment obtained in this manner was then used as a probewith which to screen the same library for human Notch clones. Theoriginal screen yielded three unique clones, hN3k, hN2K, and hN5k, allof which were shown by subsequent sequence analysis to fall in the 3'end of human Notch (FIG. 17). A second screen using the 5' end of hN3kas probe was undertaken to search for clones encompassing the 5' end ofhuman Notch. One unique clone, hN4k, was obtained from this screen, andpreliminary sequencing data indicate that it contains most of the 5' endof the gene (FIG. 17). Together, clones hN4k, hN3k and hN5k encompassabout 10 kb of the human Notch homolog, beginning early in theEGF-repeats and extending into the 3' untranslated region of the gene.All three clones are cDNA inserts in the EcoRI site of pBluescript SK⁻(Stratagene). The host E. coli strain is XL1-Blue (see Maniatis, T.,1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. A12).

The sequence of various portions of Notch contained in the cDNA cloneswas determined (by use of Sequenase®, U.S. Biochemical Corp.) and isshown in FIGS. 19A-22D (SEQ ID NO:13 through NO:25).

The complete nucleotide sequences of the human Notch cDNA contained inhN3k and hN5k was determined by the dideoxy chain termination methodusing the Sequenase® kit (U.S. Biochemical Corp.). Those nucleotidesequences encoding human Notch, in the appropriate reading frame, werereadily identified since translation in only one out of the threepossible reading frames yields a sequence which, upon comparison withthe published Drosophila Notch deduced amino acid sequence, yields asequence with a substantial degree of homology to the Drosophila Notchsequence. Since there are no introns, translation of all three possiblereading frames and comparison with Drosophila Notch was easilyaccomplished, leading to the ready identification of the coding region.The DNA and deduced protein sequences of the human Notch cDNA in hN3kand hN5k are presented in FIGS. 23A-23Q and and 24A-24G , respectively.Clone hN3k encodes a portion of a Notch polypeptide starting atapproximately the third Notch/lin-12 repeat to several amino acids shortof the carboxy-terminal amino acid. Clone hN5k encodes a portion of aNotch polypeptide starting approximately before the cdc10 region throughthe end of the polypeptide, and also contains a 3' untranslated region.

Comparing the DNA and protein sequences presented in FIGS. 23A-23Q (SEQID NO:31 and NO:32) with those in FIGS. 24A-24G (SEQ ID NO:33 and NO:34)reveals significant differences between the sequences, suggesting thathN3k and hN5k represent part of two distinct Notch-homologous genes. Ourdata thus suggest that the human genome harbors more than oneNotch-homologous gene. This is unlike Drosophila, where Notch appears tobe a single-copy gene.

Comparison of the DNA and amino acid sequences of the human Notchhomologs contained in hN3k and hN5k with the corresponding DrosophilaNotch sequences (as published in Wharton et al., 1985, Cell 43:567-581)and with the corresponding Xenopus Notch sequences (as published inCoffman et al., 1990, Science 249:1438-1441 or available from Genbank®(accession number M33874)) also revealed differences.

The amino acid sequence shown in FIGS. 23A-23Q (hN3k) was compared withthe predicted sequence of the TAN-1 polypeptide shown in FIG. 2 ofEllisen et al., August 1991, Cell 66:649-661. Some differences werefound between the deduced amino acid sequences; however, overall thehN3k Notch polypeptide sequence is 99% identical to the correspondingTAN-1 region (TAN-1 amino acids 1455 to 2506). Four differences werenoted: in the region between the third Notch/lin-12 repeat and the firstcdc10 motif, there is an arginine (hN3k) instead of an X (TAN-1 aminoacid 1763); (2) there is a proline (hN3k) instead of an X (TAN-1, aminoacid 1787); (3) there is a conservative change of an aspartic acidresidue (hN3k) instead of a glutamic acid residue (TAN-1, amino acid2495); and (4) the carboxyl-terminal region differs substantiallybetween TAN-1 amino acids 2507 and 2535.

The amino acid sequence shown in FIGS. 24A-24G (hN5k) was compared withthe predicted sequence of the TAN-1 polypeptide shown in FIG. 2 ofEllisen et al., August 1991, Cell 66:649-661. Differences were foundbetween the deduced amino acid sequences. The deduced Notch polypeptideof hN5k is 79% identical to the TAN-1 polypeptide (64% identical toDrosophila Notch) in the cdc10 region that encompasses both the cc10motif (TAN-1 amino acids 1860 to 2217) and the well-conserved flankingregions (FIG. 25). The cdc10 region covers amino acids 1860 through 2217of the TAN-1 sequence. In addition, the hN5k encoded polypeptide is 65%identical to the TAN-1 polypeptide (44% identical to Drosophila Notch)at the carboxy-terminal end of the molecule containing a PEST (proline,glutamic acid, serine, threonine)-rich region (TAN-1 amino acids 2482 to2551) (FIG. 25B). The stretch of 215 amino acids lying between theaforementioned regions is not well conserved among any of theNotch-homologous clones represented by hN3k, hN5k, and TAN-1. Neitherthe hN5k polypeptide nor Drosophila Notch shows significant levels ofamino acid identity to the other proteins in this region (e.g.,hN5k/TAN-1=24% identity; hN5k/Drosophila Notch=11% identity;TAN-1/Drosophila Notch=17% identity). In contrast, Xenopus Notch (Xotch)(SEQ ID NO:35), rat Notch (SEQ ID NO:36), and TAN-1 (SEQ ID NO:37)continue to share significant levels of sequence identity with oneanother (e.g., TAN-1/rat Notch=75% identity, TAN-1/Xenopus Notch=45%identity, rat Notch/Xenopus Notch=50% identity).

Finally, examination of the sequence of the intracellular domains of thevertebrate Notch homologs shown in FIGS. 25B-25C revealed an unexpectedfinding: all of these proteins, including hN5k, contain a putative CcNmotif, associated with nuclear targeting function, in the conservedregion following the last of the six cdc10 repeats (FIGS. 25B-25C).Although Drosophila Notch lacks such a defined motif, closer inspectionof its sequence revealed the presence of a possible bipartite nuclearlocalization sequence (Robbins et al., 1991, Cell 64:615-623), as wellas of possible CK II and cdc2 phosphorylation sites, all in relativeproximity to one another, thus possibly defining an alternative type ofCcN motif (FIGS. 25B-25C).

10.2. EXPRESSION OF HUMAN NOTCH

Expression constructs were made using the human Notch cDNA clonesdiscussed in Section 10.1 above. In the cases of hN3k and hN2k, theentire clone was excised from its vector as an EcoRI restrictionfragment and subcloned into the EcoRI restriction site of each of thethree pGEX vectors (Glutathione S-Transferase expression vectors; Smithand Johnson, 1988, Gene 7, 31-40). This allows for the expression of theNotch protein product from the subclone in the correct reading frame. Inthe case of hN5k, the clone contains two internal EcoRI restrictionsites, producing 2.6, 1.5 and 0.6 kb fragments. Both the 2.6 and the 1.5kb fragments have also been subcloned into each of the pGEX vectors.

The pGEX vector system was used to obtain expression of human Notchfusion (chimeric) proteins from the constructs described below. Thecloned Notch DNA in each case was inserted, in phase, into theappropriate pGEX vector. Each construct was then electroporated intobacteria (E. coli), and was expressed as a fusion protein containing theNotch protein sequences fused to the carboxyl terminus of glutathioneS-transferase protein. Expression of the fusion proteins was confirmedby analysis of bacterial protein extracts by polyacrylamide gelelectrophoresis, comparing protein extracts obtained from bacteriacontaining the pGEX plasmids with and without the inserted Notch DNA.The fusion proteins were soluble in aqueous solution, and were purifiedfrom bacterial lysates by affinity chromatography usingglutathione-coated agarose (since the carboxyl terminus of glutathioneS-transferase binds to glutathionine). The expressed fusion proteinswere bound by an antibody to Drosophila Notch, as assayed by Westernblotting.

The constructs used to make human Notch-glutathione S-transferase fusionproteins were as follows:

hNFP#2--PCR was used to obtain a fragment starting just before the cdc10repeats at nucleotide 192 of the hN5k insert to just before thePEST-rich region at nucleotide 1694. The DNA was then digested withBamHI and SmaI and the resulting fragment was ligated into pGEX-3. Afterexpression, the fusion protein was purified by binding to glutathioneagarose. The purified polypeptide was quantitated on a 4-15% gradientpolyacrylamide gel. The resulting fusion protein had an approximatemolecular weight of 83 kD.

hN3FP#1--The entire hN3k DNA insert (nucleotide 1 to 3235) was excisedfrom the Bluescript (SK) vector by digesting with EcoRI. The DNA wasligated into pGEX-3.

hN3FP#2--A 3' segment of hN3k DNA (nucleotide 1847 to 3235) plus some ofthe polylinker was cut out of the Bluescript (SK) vector by digestingwith XmaI. The fragment was ligated into pGEX-1.

Following purification, these fusion proteins are used to make eitherpolyclonal and/or monoclonal antibodies to human Notch.

11. DEPOSIT OF MICROORGANISMS

The following recombinant bacteria, each carrying a plasmid encoding aportion of human Notch, were deposited on May 2, 1991 with the AmericanType Culture Collection, 1201 Parklawn Drive, Rockville, Md. 20852,under the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedures.

    ______________________________________    Bacteria   carrying Plasmid   ATCC Accession No.    ______________________________________    E. coli XL1-Blue                    hN4k      68610    E. coli XL1-Blue                    hN3k      68609    E. coli XL1-Blue                    hN5k      68611    ______________________________________

The present invention is not to be limited in scope by themicroorganisms deposited or the specific embodiments described herein.Indeed, various modifications of the invention in addition to thosedescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying figures. Such modificationsare intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 37    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 77 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GluAspIleAspGluCysAspGlnGlySerProCysGluHisAsnGly    151015    IleCysValAsnThrProGlySerTyrArgCysAsnCysSerGlnGly    202530    PheThrGlyProArgCysGluThrAsnIleAsnGluCysGluSerHis    354045    ProCysGlnAsnGluGlySerCysLeuAspAspProGlyThrPheArg    505560    CysValCysMetProGlyPheThrGlyThrGlnCysGlu    657075    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 78 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    AsnAspValAspGluCysSerLeuGlyAlaAsnProCysGluHisGly    151015    GlyArgCysThrAsnThrLeuGlySerPheGlnCysAsnCysProGln    202530    GlyTyrAlaGlyProArgCysGluIleAspValAsnGluCysLeuSer    354045    AsnProCysGlnAsnAspSerThrCysLeuAspGlnIleGlyGluPhe    505560    GlnCysIleCysMetProGlyTyrGluGlyLeuTyrCysGlu    657075    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 203 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GlySerPheGluLeuArgLeuLysTyrPheSerAsnAspHisGlyArg    151015    AspAsnGluGlyArgCysCysSerGlyGluSerAspGlyAlaThrGly    202530    LysCysLeuGlySerCysLysThrArgPheArgValCysLeuLysHis    354045    TyrGlnAlaThrIleAspThrThrSerGlnCysThrTyrGlyAspVal    505560    IleThrProIleLeuGlyGluAsnSerValAsnLeuThrAspAlaGln    65707580    ArgPheGlnAsnLysGlyPheThrAsnProIleGlnPheProPheSer    859095    PheSerTrpProGlyThrPheSerLeuIleValGluAlaTrpHisAsp    100105110    ThrAsnAsnSerGlyAsnAlaArgThrAsnLysLeuLeuIleGlnArg    115120125    LeuLeuValGlnGlnValLeuGluValSerSerGluTrpLysThrAsn    130135140    LysSerGluSerGlnTyrThrSerLeuGluTyrAspPheArgValThr    145150155160    CysAspLeuAsnTyrTyrGlySerGlyCysAlaLysPheCysArgPro    165170175    ArgAspAspSerPheGlyHisSerThrCysSerGluThrGlyGluIle    180185190    IleCysLeuThrGlyTrpGlnGlyAspTyrCys    195200    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 199 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GlyAsnPheGluLeuGluIleLeuGluIleSerAsnThrAsnSerHis    151015    LeuLeuAsnGlyTyrCysCysGlyMetProAlaGluLeuArgAlaThr    202530    LysThrIleGlyCysSerProCysThrThrAlaPheArgLeuCysLeu    354045    LysGluTyrGlnThrThrGluGlnGlyAlaSerIleSerThrGlyCys    505560    SerPheGlyAsnAlaThrThrLysIleLeuGlyGlySerSerPheVal    65707580    LeuSerAspProGlyValGlyAlaIleValLeuProPheThrPheArg    859095    TrpThrLysSerPheThrLeuIleLeuGlnAlaLeuAspMetTyrAsn    100105110    ThrSerTyrProAspAlaGluArgLeuIleGluGluThrSerTyrSer    115120125    GlyValIleLeuProSerProGluTrpLysThrLeuAspHisIleGly    130135140    ArgAsnAlaArgIleThrTyrArgValArgValGlnCysAlaValThr    145150155160    TyrTyrAsnThrThrCysThrThrPheCysArgProArgAspAspGln    165170175    PheGlyHisTyrAlaCysGlySerGluGlyGlnLysLeuCysLeuAsn    180185190    GlyTrpGlnGlyValAsnCys    195    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2892 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 142..2640    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GAATTCGGAGGAATTATTCAAAACATAAACACAATAAACAATTTGAGTAGTTGCCGCACA60    CACACACACACACAGCCCGTGGATTATTACACTAAAAGCGACACTCAATCCAAAAAATCA120    GCAACAAAAACATCAATAAACATGCATTGGATTAAATGTTTATTAACAGCA171    MetHisTrpIleLysCysLeuLeuThrAla    1510    TTCATTTGCTTCACAGTCATCGTGCAGGTTCACAGTTCCGGCAGCTTT219    PheIleCysPheThrValIleValGlnValHisSerSerGlySerPhe    152025    GAGTTGCGCCTGAAGTACTTCAGCAACGATCACGGGCGGGACAACGAG267    GluLeuArgLeuLysTyrPheSerAsnAspHisGlyArgAspAsnGlu    303540    GGTCGCTGCTGCAGCGGGGAGTCGGACGGAGCGACGGGCAAGTGCCTG315    GlyArgCysCysSerGlyGluSerAspGlyAlaThrGlyLysCysLeu    455055    GGCAGCTGCAAGACGCGGTTTCGCGTCTGCCTAAAGCACTACCAGGCC363    GlySerCysLysThrArgPheArgValCysLeuLysHisTyrGlnAla    606570    ACCATCGACACCACCTCCCAGTGCACCTACGGGGACGTGATCACGCCC411    ThrIleAspThrThrSerGlnCysThrTyrGlyAspValIleThrPro    75808590    ATTCTCGGCGAGAACTCGGTCAATCTGACCGACGCCCAGCGCTTCCAG459    IleLeuGlyGluAsnSerValAsnLeuThrAspAlaGlnArgPheGln    95100105    AACAAGGGCTTCACGAATCCCATCCAGTTCCCCTTCTCGTTCTCATGG507    AsnLysGlyPheThrAsnProIleGlnPheProPheSerPheSerTrp    110115120    CCGGGTACCTTCTCGCTGATCGTCGAGGCCTGGCATGATACGAACAAT555    ProGlyThrPheSerLeuIleValGluAlaTrpHisAspThrAsnAsn    125130135    AGCGGCAATGCGCGAACCAACAAGCTCCTCATCCAGCGACTCTTGGTG603    SerGlyAsnAlaArgThrAsnLysLeuLeuIleGlnArgLeuLeuVal    140145150    CAGCAGGTACTGGAGGTGTCCTCCGAATGGAAGACGAACAAGTCGGAA651    GlnGlnValLeuGluValSerSerGluTrpLysThrAsnLysSerGlu    155160165170    TCGCAGTACACGTCGCTGGAGTACGATTTCCGTGTCACCTGCGATCTC699    SerGlnTyrThrSerLeuGluTyrAspPheArgValThrCysAspLeu    175180185    AACTACTACGGATCCGGCTGTGCCAAGTTCTGCCGGCCCCGCGACGAT747    AsnTyrTyrGlySerGlyCysAlaLysPheCysArgProArgAspAsp    190195200    TCATTTGGACACTCGACTTGCTCGGAGACGGGCGAAATTATCTGTTTG795    SerPheGlyHisSerThrCysSerGluThrGlyGluIleIleCysLeu    205210215    ACCGGATGGCAGGGCGATTACTGTCACATACCCAAATGCGCCAAAGGC843    ThrGlyTrpGlnGlyAspTyrCysHisIleProLysCysAlaLysGly    220225230    TGTGAACATGGACATTGCGACAAACCCAATCAATGCGTTTGCCAACTG891    CysGluHisGlyHisCysAspLysProAsnGlnCysValCysGlnLeu    235240245250    GGCTGGAAGGGAGCCTTGTGCAACGAGTGCGTTCTGGAACCGAACTGC939    GlyTrpLysGlyAlaLeuCysAsnGluCysValLeuGluProAsnCys    255260265    ATCCATGGCACCTGCAACAAACCCTGGACTTGCATCTGCAACGAGGGT987    IleHisGlyThrCysAsnLysProTrpThrCysIleCysAsnGluGly    270275280    TGGGGAGGCTTGTACTGCAACCAGGATCTGAACTACTGCACCAACCAC1035    TrpGlyGlyLeuTyrCysAsnGlnAspLeuAsnTyrCysThrAsnHis    285290295    AGACCCTGCAAGAATGGCGGAACCTGCTTCAACACCGGCGAGGGATTG1083    ArgProCysLysAsnGlyGlyThrCysPheAsnThrGlyGluGlyLeu    300305310    TACACATGCAAATGCGCTCCAGGATACAGTGGTGATGATTGCGAAAAT1131    TyrThrCysLysCysAlaProGlyTyrSerGlyAspAspCysGluAsn    315320325330    GAGATCTACTCCTGCGATGCCGATGTCAATCCCTGCCAGAATGGTGGT1179    GluIleTyrSerCysAspAlaAspValAsnProCysGlnAsnGlyGly    335340345    ACCTGCATCGATGAGCCGCACACAAAAACCGGCTACAAGTGTCATTGC1227    ThrCysIleAspGluProHisThrLysThrGlyTyrLysCysHisCys    350355360    GCCAACGGCTGGAGCGGAAAGATGTGCGAGGAGAAAGTGCTCACGTGT1275    AlaAsnGlyTrpSerGlyLysMetCysGluGluLysValLeuThrCys    365370375    TCGGACAAACCCTGTCATCAGGGAATCTGCCGCAACGTTCGTCCTGGC1323    SerAspLysProCysHisGlnGlyIleCysArgAsnValArgProGly    380385390    TTGGGAAGCAAGGGTCAGGGCTACCAGTGCGAATGTCCCATTGGCTAC1371    LeuGlySerLysGlyGlnGlyTyrGlnCysGluCysProIleGlyTyr    395400405410    AGCGGACCCAACTGCGATCTCCAGCTGGACAACTGCAGTCCGAATCCA1419    SerGlyProAsnCysAspLeuGlnLeuAspAsnCysSerProAsnPro    415420425    TGCATAAACGGTGGAAGCTGTCAGCCGAGCGGAAAGTGTATTTGCCCA1467    CysIleAsnGlyGlySerCysGlnProSerGlyLysCysIleCysPro    430435440    GCGGGATTTTCGGGAACGAGATGCGAGACCAACATTGACGATTGTCTT1515    AlaGlyPheSerGlyThrArgCysGluThrAsnIleAspAspCysLeu    445450455    GGCCACCAGTGCGAGAACGGAGGCACCTGCATAGATATGGTCAACCAA1563    GlyHisGlnCysGluAsnGlyGlyThrCysIleAspMetValAsnGln    460465470    TATCGCTGCCAATGCGTTCCCGGTTTCCATGGCACCCACTGTAGTAGC1611    TyrArgCysGlnCysValProGlyPheHisGlyThrHisCysSerSer    475480485490    AAAGTTGACTTGTGCCTCATCAGACCGTGTGCCAATGGAGGAACCTGC1659    LysValAspLeuCysLeuIleArgProCysAlaAsnGlyGlyThrCys    495500505    TTGAATCTCAACAACGATTACCAGTGCACCTGTCGTGCGGGATTTACT1707    LeuAsnLeuAsnAsnAspTyrGlnCysThrCysArgAlaGlyPheThr    510515520    GGCAAGGATTGCTCTGTGGACATCGATGAGTGCAGCAGTGGACCCTGT1755    GlyLysAspCysSerValAspIleAspGluCysSerSerGlyProCys    525530535    CATAACGGCGGCACTTGCATGAACCGCGTCAATTCGTTCGAATGCGTG1803    HisAsnGlyGlyThrCysMetAsnArgValAsnSerPheGluCysVal    540545550    TGTGCCAATGGTTTCAGGGGCAAGCAGTGCGATGAGGAGTCCTACGAT1851    CysAlaAsnGlyPheArgGlyLysGlnCysAspGluGluSerTyrAsp    555560565570    TCGGTGACCTTCGATGCCCACCAATATGGAGCGACCACACAAGCGAGA1899    SerValThrPheAspAlaHisGlnTyrGlyAlaThrThrGlnAlaArg    575580585    GCCGATGGTTTGACCAATGCCCAGGTAGTCCTAATTGCTGTTTTCTCC1947    AlaAspGlyLeuThrAsnAlaGlnValValLeuIleAlaValPheSer    590595600    GTTGCGATGCCTTTGGTGGCGGTTATTGCGGCGTGCGTGGTCTTCTGC1995    ValAlaMetProLeuValAlaValIleAlaAlaCysValValPheCys    605610615    ATGAAGCGCAAGCGTAAGCGTGCTCAGGAAAAGGACGACGCGGAGGCC2043    MetLysArgLysArgLysArgAlaGlnGluLysAspAspAlaGluAla    620625630    AGGAAGCAGAACGAACAGAATGCGGTGGCCACAATGCATCACAATGGC2091    ArgLysGlnAsnGluGlnAsnAlaValAlaThrMetHisHisAsnGly    635640645650    AGTGGGGTGGGTGTAGCTTTGGCTTCAGCCTCTCTGGGCGGCAAAACT2139    SerGlyValGlyValAlaLeuAlaSerAlaSerLeuGlyGlyLysThr    655660665    GGCAGCAACAGCGGTCTCACCTTCGATGGCGGCAACCCGAATATCATC2187    GlySerAsnSerGlyLeuThrPheAspGlyGlyAsnProAsnIleIle    670675680    AAAAACACCTGGGACAAGTCGGTCAACAACATTTGTGCCTCAGCAGCA2235    LysAsnThrTrpAspLysSerValAsnAsnIleCysAlaSerAlaAla    685690695    GCAGCGGCGGCGGCGGCAGCAGCGGCGGACGAGTGTCTCATGTACGGC2283    AlaAlaAlaAlaAlaAlaAlaAlaAlaAspGluCysLeuMetTyrGly    700705710    GGATATGTGGCCTCGGTGGCGGATAACAACAATGCCAACTCAGACTTT2331    GlyTyrValAlaSerValAlaAspAsnAsnAsnAlaAsnSerAspPhe    715720725730    TGTGTGGCTCCGCTACAAAGAGCCAAGTCGCAAAAGCAACTCAACACC2379    CysValAlaProLeuGlnArgAlaLysSerGlnLysGlnLeuAsnThr    735740745    GATCCCACGCTCATGCACCGCGGTTCGCCGGCAGGCAGCTCAGCCAAG2427    AspProThrLeuMetHisArgGlySerProAlaGlySerSerAlaLys    750755760    GGAGCGTCTGGCGGAGGACCGGGAGCGGCGGAGGGCAAGAGGATCTCT2475    GlyAlaSerGlyGlyGlyProGlyAlaAlaGluGlyLysArgIleSer    765770775    GTTTTAGGCGAGGGTTCCTACTGTAGCCAGCGTTGGCCCTCGTTGGCG2523    ValLeuGlyGluGlySerTyrCysSerGlnArgTrpProSerLeuAla    780785790    GCGGCGGGAGTGGCCGGAGCCTGTTCATCCCAGCTAATGGCTGCAGCT2571    AlaAlaGlyValAlaGlyAlaCysSerSerGlnLeuMetAlaAlaAla    795800805810    TCGGCAGCGGGCAGCGGAGCGGGGACGGCGCAACAGCAGCGATCCGTG2619    SerAlaAlaGlySerGlyAlaGlyThrAlaGlnGlnGlnArgSerVal    815820825    GTCTGCGGCACTCCGCATATGTAACTCCAAAAATCCGGAAGGGCTCCTGGT2670    ValCysGlyThrProHisMet    830    AAATCCGGAGAAATCCGCATGGAGGAGCTGACAGCACATACACAAAGAAAAGACTGGGTT2730    GGGTTCAAAATGTGAGAGAGACGCCAAAATGTTGTTGTTGATTGAAGCAGTTTAGTCGTC2790    ACGAAAAATGAAAAATCTGTAACAGGCATAACTCGTAAACTCCCTAAAAAATTTGTATAG2850    TAATTAGCAAAGCTGTGACCCAGCCGTTTCGATCCCGAATTC2892    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 833 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetHisTrpIleLysCysLeuLeuThrAlaPheIleCysPheThrVal    151015    IleValGlnValHisSerSerGlySerPheGluLeuArgLeuLysTyr    202530    PheSerAsnAspHisGlyArgAspAsnGluGlyArgCysCysSerGly    354045    GluSerAspGlyAlaThrGlyLysCysLeuGlySerCysLysThrArg    505560    PheArgValCysLeuLysHisTyrGlnAlaThrIleAspThrThrSer    65707580    GlnCysThrTyrGlyAspValIleThrProIleLeuGlyGluAsnSer    859095    ValAsnLeuThrAspAlaGlnArgPheGlnAsnLysGlyPheThrAsn    100105110    ProIleGlnPheProPheSerPheSerTrpProGlyThrPheSerLeu    115120125    IleValGluAlaTrpHisAspThrAsnAsnSerGlyAsnAlaArgThr    130135140    AsnLysLeuLeuIleGlnArgLeuLeuValGlnGlnValLeuGluVal    145150155160    SerSerGluTrpLysThrAsnLysSerGluSerGlnTyrThrSerLeu    165170175    GluTyrAspPheArgValThrCysAspLeuAsnTyrTyrGlySerGly    180185190    CysAlaLysPheCysArgProArgAspAspSerPheGlyHisSerThr    195200205    CysSerGluThrGlyGluIleIleCysLeuThrGlyTrpGlnGlyAsp    210215220    TyrCysHisIleProLysCysAlaLysGlyCysGluHisGlyHisCys    225230235240    AspLysProAsnGlnCysValCysGlnLeuGlyTrpLysGlyAlaLeu    245250255    CysAsnGluCysValLeuGluProAsnCysIleHisGlyThrCysAsn    260265270    LysProTrpThrCysIleCysAsnGluGlyTrpGlyGlyLeuTyrCys    275280285    AsnGlnAspLeuAsnTyrCysThrAsnHisArgProCysLysAsnGly    290295300    GlyThrCysPheAsnThrGlyGluGlyLeuTyrThrCysLysCysAla    305310315320    ProGlyTyrSerGlyAspAspCysGluAsnGluIleTyrSerCysAsp    325330335    AlaAspValAsnProCysGlnAsnGlyGlyThrCysIleAspGluPro    340345350    HisThrLysThrGlyTyrLysCysHisCysAlaAsnGlyTrpSerGly    355360365    LysMetCysGluGluLysValLeuThrCysSerAspLysProCysHis    370375380    GlnGlyIleCysArgAsnValArgProGlyLeuGlySerLysGlyGln    385390395400    GlyTyrGlnCysGluCysProIleGlyTyrSerGlyProAsnCysAsp    405410415    LeuGlnLeuAspAsnCysSerProAsnProCysIleAsnGlyGlySer    420425430    CysGlnProSerGlyLysCysIleCysProAlaGlyPheSerGlyThr    435440445    ArgCysGluThrAsnIleAspAspCysLeuGlyHisGlnCysGluAsn    450455460    GlyGlyThrCysIleAspMetValAsnGlnTyrArgCysGlnCysVal    465470475480    ProGlyPheHisGlyThrHisCysSerSerLysValAspLeuCysLeu    485490495    IleArgProCysAlaAsnGlyGlyThrCysLeuAsnLeuAsnAsnAsp    500505510    TyrGlnCysThrCysArgAlaGlyPheThrGlyLysAspCysSerVal    515520525    AspIleAspGluCysSerSerGlyProCysHisAsnGlyGlyThrCys    530535540    MetAsnArgValAsnSerPheGluCysValCysAlaAsnGlyPheArg    545550555560    GlyLysGlnCysAspGluGluSerTyrAspSerValThrPheAspAla    565570575    HisGlnTyrGlyAlaThrThrGlnAlaArgAlaAspGlyLeuThrAsn    580585590    AlaGlnValValLeuIleAlaValPheSerValAlaMetProLeuVal    595600605    AlaValIleAlaAlaCysValValPheCysMetLysArgLysArgLys    610615620    ArgAlaGlnGluLysAspAspAlaGluAlaArgLysGlnAsnGluGln    625630635640    AsnAlaValAlaThrMetHisHisAsnGlySerGlyValGlyValAla    645650655    LeuAlaSerAlaSerLeuGlyGlyLysThrGlySerAsnSerGlyLeu    660665670    ThrPheAspGlyGlyAsnProAsnIleIleLysAsnThrTrpAspLys    675680685    SerValAsnAsnIleCysAlaSerAlaAlaAlaAlaAlaAlaAlaAla    690695700    AlaAlaAlaAspGluCysLeuMetTyrGlyGlyTyrValAlaSerVal    705710715720    AlaAspAsnAsnAsnAlaAsnSerAspPheCysValAlaProLeuGln    725730735    ArgAlaLysSerGlnLysGlnLeuAsnThrAspProThrLeuMetHis    740745750    ArgGlySerProAlaGlySerSerAlaLysGlyAlaSerGlyGlyGly    755760765    ProGlyAlaAlaGluGlyLysArgIleSerValLeuGlyGluGlySer    770775780    TyrCysSerGlnArgTrpProSerLeuAlaAlaAlaGlyValAlaGly    785790795800    AlaCysSerSerGlnLeuMetAlaAlaAlaSerAlaAlaGlySerGly    805810815    AlaGlyThrAlaGlnGlnGlnArgSerValValCysGlyThrProHis    820825830    Met    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1067 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GATCTACTACGAGGAGGTTAAGGAGAGCTATGTGGGCGAGCGACGCGAATACGATCCCCA60    CATCACCGATCCCAGGGTCACACGCATGAAGATGGCCGGCCTGAAGCCCAACTCCAAATA120    CCGCATCTCCATCACTGCCACCACGAAAATGGGCGAGGGATCTGAACACTATATCGAAAA180    GACCACGCTCAAGGATGCCGTCAATGTGGCCCCTGCCACGCCATCTTTCTCCTGGGAGCA240    ACTGCCATCCGACAATGGACTAGCCAAGTTCCGCATCAACTGGCTGCCAAGTACCGAGGG300    TCATCCAGGCACTCACTTCTTTACGATGCACAGGATCAAGGGCGAAACCCAATGGATACG360    CGAGAATGAGGAAAAGAACTCCGATTACCAGGAGGTCGGTGGCTTAGATCCGGAGACCGC420    CTACGAGTTCCGCGTGGTGTCCGTGGATGGCCACTTTAACACGGAGAGTGCCACGCAGGA480    GATCGACACGAACACCGTTGAGGGACCAATAATGGTGGCCAACGAGACGGTGGCCAATGC540    CGGATGGTTCATTGGCATGATGCTGGCCCTGGCCTTCATCATCATCCTCTTCATCATCAT600    CTGCATTATCCGACGCAATCGGGGCGGAAAGTACGATGTCCACGATCGGGAGCTGGCCAA660    CGGCCGGCGGGATTATCCCGAAGAGGGCGGATTCCACGAGTACTCGCAACCGTTGGATAA720    CAAGAGCGCTGGTCGCCAATCCGTGAGTTCAGCGAACAAACCGGGCGTGGAAAGCGATAC780    TGATTCGATGGCCGAATACGGTGATGGCGATACAGGACAATTTACCGAGGATGGCTCCTT840    CATTGGCCAATATGTTCCTGGAAAGCTCCAACCGCCGGTTAGCCCACAGCCACTGAACAA900    TTCCGCTGCGGCGCATCAGGCGGCGCCAACTGCCGGAGGATCGGGAGCAGCCGGATCGGC960    AGCAGCAGCCGGAGCATCGGGTGGAGCATCGTCCGCCGGAGGAGCAGCTGCCAGCAATGG1020    AGGAGCTGCAGCCGGAGCCGTGGCCACCTACGTCTAAGCTTGGTACC1067    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1320 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 442..1320    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    CCGAGTCGAGCGCCGTGCTTCGAGCGGTGATGAGCCCCTTTTCTGTCAACGCTAAAGATC60    TACAAAACATCAGCGCCTATCAAGTGGAAGTGTCAAGTGTGAACAAAACAAAAACGAGAG120    AAGCACATACTAAGGTCCATATAAATAATAAATAATAATTGTGTGTGATAACAACATTAT180    CCAAACAAAACCAAACAAAACGAAGGCAAAGTGGAGAAAATGATACAGCATCCAGAGTAC240    GGCCGTTATTCAGCTATCCAGAGCAAGTGTAGTGTGGCAAAATAGAAACAAACAAAGGCA300    CCAAAATCTGCATACATGGGCTAATTAAGGCTGCCCAGCGAATTTACATTTGTGTGGTGC360    CAATCCAGAGTGAATCCGAAACAAACTCCATCTAGATCGCCAACCAGCATCACGCTCGCA420    AACGCCCCCAGAATGTACAAAATGTTTAGGAAACATTTTCGGCGAAAACCA471    MetPheArgLysHisPheArgArgLysPro    1510    GCTACGTCGTCGTCGTTGGAGTCAACAATAGAATCAGCAGACAGCCTG519    AlaThrSerSerSerLeuGluSerThrIleGluSerAlaAspSerLeu    152025    GGAATGTCCAAGAAGACGGCGACAAAAAGGCAGCGTCCGAGGCATCGG567    GlyMetSerLysLysThrAlaThrLysArgGlnArgProArgHisArg    303540    GTACCCAAAATCGCGACCCTGCCATCGACGATCCGCGATTGTCGATCA615    ValProLysIleAlaThrLeuProSerThrIleArgAspCysArgSer    455055    TTAAAGTCTGCCTGCAACTTAATTGCTTTAATTTTAATACTGTTAGTC663    LeuLysSerAlaCysAsnLeuIleAlaLeuIleLeuIleLeuLeuVal    606570    CATAAGATATCCGCAGCTGGTAACTTCGAGCTGGAAATATTAGAAATC711    HisLysIleSerAlaAlaGlyAsnPheGluLeuGluIleLeuGluIle    75808590    TCAAATACCAACAGCCATCTACTCAACGGCTATTGCTGCGGCATGCCA759    SerAsnThrAsnSerHisLeuLeuAsnGlyTyrCysCysGlyMetPro    95100105    GCGGAACTTAGGGCCACCAAGACGATAGGCTGCTCGCCATGCACGACG807    AlaGluLeuArgAlaThrLysThrIleGlyCysSerProCysThrThr    110115120    GCATTCCGGCTGTGCCTGAAGGAGTACCAGACCACGGAGCAGGGTGCC855    AlaPheArgLeuCysLeuLysGluTyrGlnThrThrGluGlnGlyAla    125130135    AGCATATCCACGGGCTGTTCGTTTGGCAACGCCACCACCAAGATACTG903    SerIleSerThrGlyCysSerPheGlyAsnAlaThrThrLysIleLeu    140145150    GGTGGCTCCAGCTTTGTGCTCAGCGATCCGGGTGTGGGAGCCATTGTG951    GlyGlySerSerPheValLeuSerAspProGlyValGlyAlaIleVal    155160165170    CTGCCCTTTACGTTTCGTTGGACGAAGTCGTTTACGCTGATACTGCAG999    LeuProPheThrPheArgTrpThrLysSerPheThrLeuIleLeuGln    175180185    GCGTTGGATATGTACAACACATCCTATCCAGATGCGGAGAGGTTAATT1047    AlaLeuAspMetTyrAsnThrSerTyrProAspAlaGluArgLeuIle    190195200    GAGGAAACATCATACTCGGGCGTGATACTGCCGTCGCCGGAGTGGAAG1095    GluGluThrSerTyrSerGlyValIleLeuProSerProGluTrpLys    205210215    ACGCTGGACCACATCGGGCGGAACGCGCGGATCACCTACCGTGTCCGG1143    ThrLeuAspHisIleGlyArgAsnAlaArgIleThrTyrArgValArg    220225230    GTGCAATGCGCCGTTACCTACTACAACACGACCTGCACGACCTTCTGC1191    ValGlnCysAlaValThrTyrTyrAsnThrThrCysThrThrPheCys    235240245250    CGTCCGCGGGACGATCAGTTCGGTCACTACGCCTGCGGCTCCGAGGGT1239    ArgProArgAspAspGlnPheGlyHisTyrAlaCysGlySerGluGly    255260265    CAGAAGCTCTGCCTGAATGGCTGGCAGGGCGTCAACTGCGAGGAGGCC1287    GlnLysLeuCysLeuAsnGlyTrpGlnGlyValAsnCysGluGluAla    270275280    ATATGCAAGGCGGGCTGCGACCCCGTCCACGGC1320    IleCysLysAlaGlyCysAspProValHisGly    285290    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 293 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    MetPheArgLysHisPheArgArgLysProAlaThrSerSerSerLeu    151015    GluSerThrIleGluSerAlaAspSerLeuGlyMetSerLysLysThr    202530    AlaThrLysArgGlnArgProArgHisArgValProLysIleAlaThr    354045    LeuProSerThrIleArgAspCysArgSerLeuLysSerAlaCysAsn    505560    LeuIleAlaLeuIleLeuIleLeuLeuValHisLysIleSerAlaAla    65707580    GlyAsnPheGluLeuGluIleLeuGluIleSerAsnThrAsnSerHis    859095    LeuLeuAsnGlyTyrCysCysGlyMetProAlaGluLeuArgAlaThr    100105110    LysThrIleGlyCysSerProCysThrThrAlaPheArgLeuCysLeu    115120125    LysGluTyrGlnThrThrGluGlnGlyAlaSerIleSerThrGlyCys    130135140    SerPheGlyAsnAlaThrThrLysIleLeuGlyGlySerSerPheVal    145150155160    LeuSerAspProGlyValGlyAlaIleValLeuProPheThrPheArg    165170175    TrpThrLysSerPheThrLeuIleLeuGlnAlaLeuAspMetTyrAsn    180185190    ThrSerTyrProAspAlaGluArgLeuIleGluGluThrSerTyrSer    195200205    GlyValIleLeuProSerProGluTrpLysThrLeuAspHisIleGly    210215220    ArgAsnAlaArgIleThrTyrArgValArgValGlnCysAlaValThr    225230235240    TyrTyrAsnThrThrCysThrThrPheCysArgProArgAspAspGln    245250255    PheGlyHisTyrAlaCysGlySerGluGlyGlnLysLeuCysLeuAsn    260265270    GlyTrpGlnGlyValAsnCysGluGluAlaIleCysLysAlaGlyCys    275280285    AspProValHisGly    290    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 6    (D) OTHER INFORMATION: /mod.sub.-- base= i    /label= N    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 12    (D) OTHER INFORMATION: /mod.sub.-- base= i    /label= N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GAYGCNAAYGTNCARGAYAAYATGGG26    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 3    (D) OTHER INFORMATION: /mod.sub.-- base= i    /label= N    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 12    (D) OTHER INFORMATION: /mod.sub.-- base= i    /label= N    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 18    (D) OTHER INFORMATION: /mod.sub.-- base= i    /label= N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    ATNARRTCYTCNACCATNCCYTCDA25    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 12    (D) OTHER INFORMATION: /mod.sub.-- base= i    /label= N    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 18    (D) OTHER INFORMATION: /mod.sub.-- base= i    /label= N    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 21    (D) OTHER INFORMATION: /mod.sub.-- base= i    /label= N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    TCCATRTGRTCNGTDATNTCNCKRTT26    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 267 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    CGGTGGACTTCCTTCGTGTATTGGTGGGAGCCCTCGGGAACGGGGGGTAACACTGAAAGG60    TCGAGTACCCATTTCCGTCATAACGGGTTGGTCGCCCCCTAGGGGTCGGAGTCAGGTGGA120    CGGGAGGTCGACAACGCCCGGGGGACGGGTGGTACATGGTGTAAGGTCTTTACCGGACCG180    GGCAAACGGGTCACACCGAAAGGGGTGAACGGTAACTACGGGGTCGTCCTGCCCGTCCAT240    CGAGTCTGGTAAGAGGGTCGCCTTAAG267    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 574 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GAATTCCTTCCATTATACGTGACTTTTCTGAAACTGTAGCCACCCTAGTGTCTCTAACTC60    CCTCTGGAGTTTGTCAGCTTTGGTCTTTTCAAAGAGCAGGCTCTCTTCAAGCTCCTTAAT120    GCGGGCATGCTCCAGTTTGGTCTGCGTCTCAAGATCACCTTTGGTAATTGATTCTTCTTC180    AACCCGGAACTGAAGGCTGGCTCTCACCCTCTAGGCAGAGCAGGAATTCCGAGGTGGATG240    TGTTAGATGTGAATGTCCGTGGCCCAGATGGCTGCACCCCATTGATGTTGGCTTCTCTCC300    GAGGAGGCAGCTCAGATTTGAGTGATGAAGATGAAGATGCAGAGGACTGTTCTGCTAACA360    TCATCACAGACTTGGTCTACCAGGGTGCCAGCCTCCAGNCCAGACAGACCGGACTGGTGA420    GATGGCCCTGCACCTTGCAGCCCGCTACTCACGGGCTGATGCTGCCAAGCGTCTCCTGGA480    TGCAGGTGCAGATGCCAATGCCCAGGACAACATGGGCCGCTGTCCACTCCATGCTGCAGT540    GGCACGTGATGCCAAGGTGTATTCAGATCTGTTA574    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 295 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    TCCAGATTCTGATTCGCAACCGAGTAACTGATCTAGATGCCAGGATGAATGATGGTACTA60    CACCCCTGATCCTGGCTGCCCGCCTGGCTGTGGAGGGAATGGTGGCAGAACTGATCAACT120    GCCAAGCGGATGTGAATGCAGTGGATGACCATGGAAAATCTGCTCTTCACTGGGCAGCTG180    CTGTCAATAATGTGGAGGCAACTCTTTTGTTGTTGAAAAATGGGGCCAACCGAGACATGC240    AGGACAACAAGGAAGAGACACCTCTGTTTCTTGCTGCCCGGGAGGAGCTATAAGC295    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 333 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GAGTCGTCGGTCGCCGGTGGACCCGGCCTCGAAGGACTCACCTCTCGGCTCGGTCCGTCT60    GCACGTCATGACCCGGGGTCGTCGGACCGCCACGTGTGATAAGACGGGGTCCTCTCGGGG120    CGGGACGGGTGCAGCGACGGTAGGAGCGACCAGGGTGGGCACTGGCGTCGGGTCAAGGAC180    TGCGGGGGGAGCGTCGTGTCGATGAGGAGCGGACACCTGTTGTGGGGGTCGGTGGTCGAT240    GTCCACGGACAAGGACATTACCATTACTAGGCTAGAAGCCTAGGAAGATTTCCGAGTAGT300    TAAAACTAGCTTCGAGGGCTGAGTACCCTTAAG333    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 582 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    GAATTCCTGCCAGGAGGACGCGGGCAACAAGGTCTGCAGCCTGCAGTGCAACAACCACGC60    GTGCGGCTGGGACGGCGGTGACTGCTCCCTCAACTTCACAATGACCCCTGGAAGAACTGC120    ACGCAGTCTCTGCAGTGCTGGAAGTACTTCAGTGACGGCCACTGTGACAGCCAGTGCAAC180    TCAGCCGGCTGCCTCTTCGACGGCTTTGACTGCCAGCGGCGGAAGGCCAGTTGCAACCCC240    CTGTACGACCAGTACTGCAAGGACCACTTCAGCGACGGGCACTGCGACCAGGGCTGCAAC300    AGCGCGGAGTNCAGNTGGGACGGGCTGGACTGTGCGGCAGTGTACCCGAGAGCTGGCGGC360    GCACGCTGGTGGTGGTGGTGCTGATGCCGCCGGAGCAGCTGCGCAACAGCTCCTTCCACT420    TCCTGCGGGACGTCAGCCGCGTGCTGCACACCAACGTGTCTTCAAGCGTGACGCACACGG480    CCAGCAGATGATGTTCCCCTACTACGGCCGCGAGGAGGAGCTGCGCAAGCCCCATCAAGC540    GTGCCGCCGAGGGCTGGGCCGCACCTGACGCCTGCTGGGCCA582    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 150 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    TCAGCCGAGTGCTGCACACCAACGTGTCTTCAAGCGTGACGCACACGGCCAGCAGATGAT60    GTTCCCCTACTACGGCCGCGAGGAGGAGCTGCGCAAGCCCCATCAAGCGTGCCGCCGAGG120    GCTGGGCCGCACCTGACGCCTGCTGGGCCA150    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 247 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    TGGACCCGGCCTCGAAGGACTCACCTCTCGGCTCGGTCCGTCTGCACGTCGTGACCCGGG60    GTCGTCGACCGCCACGTGTGATAAGACGGGGTCCTCTCGGGGCGGGACGGGTGCAGCGAC120    GGTAGGAGCGACCAGGGTGGGCACTGGCGTCGGGTCAAGGACTGCGGGGGGAGCGTCGTG180    TCGATGAGGAGCGGACACCTGTTGTGGGGGTCGGTGGTCGATGTCCACGGACAAGGACAT240    TACCATT247    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 248 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    GAATTCCATTCAGGAGGAAAGGGTGGGGAGAGAAGCAGGCACCCACTTTCCCGTGGCTGG60    ACTCGTTCCCAGGTGGCTCCACCGGCAGCTGTGACCGCCGCAGGTGGGGGCGGAGTGCCA120    TTCAGAAAATTCCAGAAAAGCCCTACCCCAACTCGGACGGCAACGTCACACCCGTGGGTA180    GCAACTGGCACACAAACAGCCAGCGTGTCTGGGGCACGGGGGGATGGCACCCCCTGCAGG240    CAGAGCTG248    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 323 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    TACGTATCTCGAGCACAGACAGCTGACGTACACTTTTNNAGTGCGAGGGACATTCGTCCG60    ACCAGTACGAACATTTAGGCTCAGTACGGTAGGTCCATGGCCAAGACTAGGAGACGTAGG120    GAGCTACAGGTCCCGCTCGCTAAACTCGGACCACTGAAACCTCCGGTCGACAGTCGGTAA180    GCGAACAAGAGGGCCAGATCTTAGAGAAGGTGTCGCGGCGAGACTCGGGCTCGGGTCAGG240    CGGCCTTAAGGACGTCGGGCCCNNNAGGTGATCAAGATCTCGNCNCGGCGGGCGCCACCT300    CGAGGNCGAAAACAAGGGAAATC323    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 330 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    GAATTCCGAGGTGGATGTGTTAGATGTGAATGTCCGTGGCCCAGATGGCTGCACCCCATT60    GATGTTGGCTTCTCTCCGAGGAGGCAGCTCAGATTTGAGTGATGAAGATGAAGATGCAGA120    GGACTCTTCTGCTAACATCATCACAGACTTGGTCTTACCAGGGTGCCAGCCTTCCAGGCC180    CAAGAACAGACCGGACTTGGTGAGATGGCCCTGCACCTTGCAGCCCGCTACTACGGGCTG240    ATGCTGCCAAGGTTCTGGATGCAGGTGCAGATGCCAATGCCCAGGACAACATGGGCCGCT300    GTCCACTCCATGCTGCAGTGGCACTGATGC330    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 167 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    TTCAAACAAGTAAGAGACGAAATAGAGAGGTACACCGTTGTAAGACAGTCGGAGAAAGTA60    TCACACGTTTGTAAAATAGTAAGATTTACCACTGAGAGACGGGAACGTGGGTAAATAATA120    AGTGTCCTACCCCTCTTGGATAGACGTACCTGGGAGTGGTAGGAGAC167    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 225 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    AGGATGAATGATGGTACTACACCCCTGATCCTGGCTGCCCGCCTGGCTGTGGAGGGAATG60    GTGGCAGAACTGATCAACTGCCAAGCGGATGTGAATGCAGTGGATGACCATGGAAAATCT120    GCTCTTCACTGGGCAGCTGCTGTCAATAATGTGGAGGCAACTCTTTTGTTGTTGAAAAAT180    GGGGCCAACCGAGACATGCAGGACAACAAGGAAGAGACACCTCTG225    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 121 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    TTCAAACAAGTAAGAGACGAAATAGAGAGGTACACCGTTGTAAGACAGTCGGAGAAAGTA60    TCACACGTTTGTAAAATAGTAAGATTTACCACTGAGAGACGGGAACGTGGGTAAATAATA120    A121    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    ACTTCAGCAACGATCACGGG20    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    TTGGGTATGTGACAGTAATCG21    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    TTAAGTTAACTTAA14    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    GGAAGATCTTCC12    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    ArgLysIlePhe    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3234 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..3234    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    TGCCAGGAGGACGCGGGCAACAAGGTCTGCAGCCTGCAGTGCAACAAC48    CysGlnGluAspAlaGlyAsnLysValCysSerLeuGlnCysAsnAsn    151015    CACGCGTGCGGCTGGGACGGCGGTGACTGCTCCCTCAACTTCAATGAC96    HisAlaCysGlyTrpAspGlyGlyAspCysSerLeuAsnPheAsnAsp    202530    CCCTGGAAGAACTGCACGCAGTCTCTGCAGTGCTGGAAGTACTTCAGT144    ProTrpLysAsnCysThrGlnSerLeuGlnCysTrpLysTyrPheSer    354045    GACGGCCACTGTGACAGCCAGTGCAACTCAGCCGGCTGCCTCTTCGAC192    AspGlyHisCysAspSerGlnCysAsnSerAlaGlyCysLeuPheAsp    505560    GGCTTTGACTGCCAGCGTGCGGAAGGCCAGTGCAACCCCCTGTACGAC240    GlyPheAspCysGlnArgAlaGluGlyGlnCysAsnProLeuTyrAsp    65707580    CAGTACTGCAAGGACCACTTCAGCGACGGGCACTGCGACCAGGGCTGC288    GlnTyrCysLysAspHisPheSerAspGlyHisCysAspGlnGlyCys    859095    AACAGCGCGGAGTGCGAGTGGGACGGGCTGGACTGTGCGGAGCATGTA336    AsnSerAlaGluCysGluTrpAspGlyLeuAspCysAlaGluHisVal    100105110    CCCGAGAGGCTGGCGGCCGGCACGCTGGTGGTGGTGGTGCTGATGCCG384    ProGluArgLeuAlaAlaGlyThrLeuValValValValLeuMetPro    115120125    CCGGAGCAGCTGCGCAACAGCTCCTTCCACTTCCTGCGGGAGCTCAGC432    ProGluGlnLeuArgAsnSerSerPheHisPheLeuArgGluLeuSer    130135140    CGCGTGCTGCACACCAACGTGGTCTTCAAGCGTGACGCACACGGCCAG480    ArgValLeuHisThrAsnValValPheLysArgAspAlaHisGlyGln    145150155160    CAGATGATCTTCCCCTACTACGGCCGCGAGGAGGAGCTGCGCAAGCAC528    GlnMetIlePheProTyrTyrGlyArgGluGluGluLeuArgLysHis    165170175    CCCATCAAGCGTGCCGCCGAGGGCTGGGCCGCACCTGACGCCCTGCTG576    ProIleLysArgAlaAlaGluGlyTrpAlaAlaProAspAlaLeuLeu    180185190    GGCCAGGTGAAGGCCTCGCTGCTCCCTGGTGGCAGCGAGGGTGGGCGG624    GlyGlnValLysAlaSerLeuLeuProGlyGlySerGluGlyGlyArg    195200205    CGGCGGAGGGAGCTGGACCCCATGGACGTCCGCGGCTCCATCGTCTAC672    ArgArgArgGluLeuAspProMetAspValArgGlySerIleValTyr    210215220    CTGGAGATTGACAACCGGCAGTGTGTGCAGGCCTCCTCGCAGTGCTTC720    LeuGluIleAspAsnArgGlnCysValGlnAlaSerSerGlnCysPhe    225230235240    CAGAGTGCCACCGACGTGGCCGCATTCCTGGGAGCGCTCGCCTCGCTG768    GlnSerAlaThrAspValAlaAlaPheLeuGlyAlaLeuAlaSerLeu    245250255    GGCAGCCTCAACATCCCCTACAAGATCGAGGCCGTGCAGAGTGAGACC816    GlySerLeuAsnIleProTyrLysIleGluAlaValGlnSerGluThr    260265270    GTGGAGCCGCCCCCGCCGGCGCAGCTGCACTTCATGTACGTGGCGGCG864    ValGluProProProProAlaGlnLeuHisPheMetTyrValAlaAla    275280285    GCCGCCTTTGTGCTTCTGTTCTTCGTGGGCTGCGGGGTGCTGCTGTCC912    AlaAlaPheValLeuLeuPhePheValGlyCysGlyValLeuLeuSer    290295300    CGCAAGCGCCGGCGGCAGCATGGCCAGCTCTGGTTCCCTGAGGGCTTC960    ArgLysArgArgArgGlnHisGlyGlnLeuTrpPheProGluGlyPhe    305310315320    AAAGTGTCTGAGGCCAGCAAGAAGAAGCGGCGGGAGCCCCTCGGCGAG1008    LysValSerGluAlaSerLysLysLysArgArgGluProLeuGlyGlu    325330335    GACTCCGTGGGCCTCAAGCCCCTGAAGAACGCTTCAGACGGTGCCCTC1056    AspSerValGlyLeuLysProLeuLysAsnAlaSerAspGlyAlaLeu    340345350    ATGGACGACAACCAGAATGAGTGGGGGGACGAGGACCTGGAGACCAAG1104    MetAspAspAsnGlnAsnGluTrpGlyAspGluAspLeuGluThrLys    355360365    AAGTTCCGGTTCGAGGAGCCCGTGGTTCTGCCTGACCTGGACGACCAG1152    LysPheArgPheGluGluProValValLeuProAspLeuAspAspGln    370375380    ACAGACCACCGGCAGTGGACTCAGCAGCACCTGGATGCCGCTGACCTG1200    ThrAspHisArgGlnTrpThrGlnGlnHisLeuAspAlaAlaAspLeu    385390395400    CGCATGTCTGCCATGGCCCCCACACCGCCCCAGGGTGAGGTTGACGCC1248    ArgMetSerAlaMetAlaProThrProProGlnGlyGluValAspAla    405410415    GACTGCATGGACGTCAATGTCCGCGGGCCTGATGGCTTCACCCCGCTC1296    AspCysMetAspValAsnValArgGlyProAspGlyPheThrProLeu    420425430    ATGATCGCCTCCTGCAGCGGGGGCGGCCTGGAGACGGGCAACAGCGAG1344    MetIleAlaSerCysSerGlyGlyGlyLeuGluThrGlyAsnSerGlu    435440445    GAAGAGGAGGACGCGCCGGCCGTCATCTCCGACTTCATCTACCAGGGC1392    GluGluGluAspAlaProAlaValIleSerAspPheIleTyrGlnGly    450455460    GCCAGCCTGCACAACCAGACAGACCGCACGGGCGAGACCGCCTTGCAC1440    AlaSerLeuHisAsnGlnThrAspArgThrGlyGluThrAlaLeuHis    465470475480    CTGGCCGCCCGCTACTCACGCTCTGATGCCGCCAAGCGCCTGCTGGAG1488    LeuAlaAlaArgTyrSerArgSerAspAlaAlaLysArgLeuLeuGlu    485490495    GCCAGCGCAGATGCCAACATCCAGGACAACATGGGCCGCACCCCGCTG1536    AlaSerAlaAspAlaAsnIleGlnAspAsnMetGlyArgThrProLeu    500505510    CATGCGGCTGTGTCTGCCGACGCACAAGGTGTCTTCCAGATCCTGATC1584    HisAlaAlaValSerAlaAspAlaGlnGlyValPheGlnIleLeuIle    515520525    CGGAACCGAGCCACAGACCTGGATGCCCGCATGCATGATGGCACGACG1632    ArgAsnArgAlaThrAspLeuAspAlaArgMetHisAspGlyThrThr    530535540    CCACTGATCCTGGCTGCCCGCCTGGCCGTGGAGGGCATGCTGGAGGAC1680    ProLeuIleLeuAlaAlaArgLeuAlaValGluGlyMetLeuGluAsp    545550555560    CTCATCAACTCACACGCCGACGTCAACGCCGTAGATGACCTGGGCAAG1728    LeuIleAsnSerHisAlaAspValAsnAlaValAspAspLeuGlyLys    565570575    TCCGCCCTGCACTGGGCCGCCGCCGTGAACAATGTGGATGCCGCAGTT1776    SerAlaLeuHisTrpAlaAlaAlaValAsnAsnValAspAlaAlaVal    580585590    GTGCTCCTGAAGAACGGGGCTAACAAAGATATGCAGAACAACAGGGAG1824    ValLeuLeuLysAsnGlyAlaAsnLysAspMetGlnAsnAsnArgGlu    595600605    GAGACACCCCTGTTTCTGGCCGCCCGGGAGGGCAGCTACGAGACCGCC1872    GluThrProLeuPheLeuAlaAlaArgGluGlySerTyrGluThrAla    610615620    AAGGTGCTGCTGGACCACTTTGCCAACCGGGACATCACGGATCATATG1920    LysValLeuLeuAspHisPheAlaAsnArgAspIleThrAspHisMet    625630635640    GACCGCCTGCCGCGCGACATCGCACAGGAGCGCATGCATCACGACATC1968    AspArgLeuProArgAspIleAlaGlnGluArgMetHisHisAspIle    645650655    GTGAGGCTGCTGGACGAGTACAACCTGGTGCGCAGCCCGCAGCTGCAC2016    ValArgLeuLeuAspGluTyrAsnLeuValArgSerProGlnLeuHis    660665670    GGAGCCCCGCTGGGGGGCACGCCCACCCTGTCGCCCCCGCTCTGCTCG2064    GlyAlaProLeuGlyGlyThrProThrLeuSerProProLeuCysSer    675680685    CCCAACGGCTACCTGGGCAGCCTCAAGCCCGGCGTGCAGGGCAAGAAG2112    ProAsnGlyTyrLeuGlySerLeuLysProGlyValGlnGlyLysLys    690695700    GTCCGCAAGCCCAGCAGCAAAGGCCTGGCCTGTGGAAGCAAGGAGGCC2160    ValArgLysProSerSerLysGlyLeuAlaCysGlySerLysGluAla    705710715720    AAGGACCTCAAGGCACGGAGGAAGAAGTCCCAGGATGGCAAGGGCTGC2208    LysAspLeuLysAlaArgArgLysLysSerGlnAspGlyLysGlyCys    725730735    CTGCTGGACAGCTCCGGCATGCTCTCGCCCGTGGACTCCCTGGAGTCA2256    LeuLeuAspSerSerGlyMetLeuSerProValAspSerLeuGluSer    740745750    CCCCATGGCTACCTGTCAGACGTGGCCTCGCCGCCACTGCTGCCCTCC2304    ProHisGlyTyrLeuSerAspValAlaSerProProLeuLeuProSer    755760765    CCGTTCCAGCAGTCTCCGTCCGTGCCCCTCAACCACCTGCCTGGGATG2352    ProPheGlnGlnSerProSerValProLeuAsnHisLeuProGlyMet    770775780    CCCGACACCCACCTGGGCATCGGGCACCTGAACGTGGCGGCCAAGCCC2400    ProAspThrHisLeuGlyIleGlyHisLeuAsnValAlaAlaLysPro    785790795800    GAGATGGCGGCGCTGGGTGGGGGCGGCCGGCTGGCCTTTGAGACTGGC2448    GluMetAlaAlaLeuGlyGlyGlyGlyArgLeuAlaPheGluThrGly    805810815    CCACCTCGTCTCTCCCACCTGCCTGTGGCCTCTGGCACCAGCACCGTC2496    ProProArgLeuSerHisLeuProValAlaSerGlyThrSerThrVal    820825830    CTGGGCTCCAGCAGCGGAGGGGCCCTGAATTTCACTGTGGGCGGGTCC2544    LeuGlySerSerSerGlyGlyAlaLeuAsnPheThrValGlyGlySer    835840845    ACCAGTTTGAATGGTCAATGCGAGTGGCTGTCCCGGCTGCAGAGCGGC2592    ThrSerLeuAsnGlyGlnCysGluTrpLeuSerArgLeuGlnSerGly    850855860    ATGGTGCCGAACCAATACAACCCTCTGCGGGGGAGTGTGGCACCAGGC2640    MetValProAsnGlnTyrAsnProLeuArgGlySerValAlaProGly    865870875880    CCCCTGAGCACACAGGCCCCCTCCCTGCAGCATGGCATGGTAGGCCCG2688    ProLeuSerThrGlnAlaProSerLeuGlnHisGlyMetValGlyPro    885890895    CTGCACAGTAGCCTTGCTGCCAGCGCCCTGTCCCAGATGATGAGCTAC2736    LeuHisSerSerLeuAlaAlaSerAlaLeuSerGlnMetMetSerTyr    900905910    CAGGGCCTGCCCAGCACCCGGCTGGCCACCCAGCCTCACCTGGTGCAG2784    GlnGlyLeuProSerThrArgLeuAlaThrGlnProHisLeuValGln    915920925    ACCCAGCAGGTGCAGCCACAAAACTTACAGATGCAGCAGCAGAACCTG2832    ThrGlnGlnValGlnProGlnAsnLeuGlnMetGlnGlnGlnAsnLeu    930935940    CAGCCAGCAAACATCCAGCAGCAGCAAAGCCTGCAGCCGCCACCACCA2880    GlnProAlaAsnIleGlnGlnGlnGlnSerLeuGlnProProProPro    945950955960    CCACCACAGCCGCACCTTGGCGTGAGCTCAGCAGCCAGCGGCCACCTG2928    ProProGlnProHisLeuGlyValSerSerAlaAlaSerGlyHisLeu    965970975    GGCCGGAGCTTCCTGAGTGGAGAGCCGAGCCAGGCAGACGTGCAGCCA2976    GlyArgSerPheLeuSerGlyGluProSerGlnAlaAspValGlnPro    980985990    CTGGGCCCCAGCAGCCTGGCGGTGCACACTATTCTGCCCCAGGAGAGC3024    LeuGlyProSerSerLeuAlaValHisThrIleLeuProGlnGluSer    99510001005    CCCGCCCTGCCCACGTCGCTGCCATCCTCGCTGGTCCCACCCGTGACC3072    ProAlaLeuProThrSerLeuProSerSerLeuValProProValThr    101010151020    GCAGCCCAGTTCCTGACGCCCCCCTCGCAGCACAGCTACTCCTCGCCT3120    AlaAlaGlnPheLeuThrProProSerGlnHisSerTyrSerSerPro    1025103010351040    GTGGACAACACCCCCAGCCACCAGCTACAGGTGCCTGTTCCTGTAATG3168    ValAspAsnThrProSerHisGlnLeuGlnValProValProValMet    104510501055    GTAATGATCCGATCTTCGGATCCTTCTAAAGGCTCATCAATTTTGATC3216    ValMetIleArgSerSerAspProSerLysGlySerSerIleLeuIle    106010651070    GAAGCTCCCGACTCATGG3234    GluAlaProAspSerTrp    1075    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1078 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    CysGlnGluAspAlaGlyAsnLysValCysSerLeuGlnCysAsnAsn    151015    HisAlaCysGlyTrpAspGlyGlyAspCysSerLeuAsnPheAsnAsp    202530    ProTrpLysAsnCysThrGlnSerLeuGlnCysTrpLysTyrPheSer    354045    AspGlyHisCysAspSerGlnCysAsnSerAlaGlyCysLeuPheAsp    505560    GlyPheAspCysGlnArgAlaGluGlyGlnCysAsnProLeuTyrAsp    65707580    GlnTyrCysLysAspHisPheSerAspGlyHisCysAspGlnGlyCys    859095    AsnSerAlaGluCysGluTrpAspGlyLeuAspCysAlaGluHisVal    100105110    ProGluArgLeuAlaAlaGlyThrLeuValValValValLeuMetPro    115120125    ProGluGlnLeuArgAsnSerSerPheHisPheLeuArgGluLeuSer    130135140    ArgValLeuHisThrAsnValValPheLysArgAspAlaHisGlyGln    145150155160    GlnMetIlePheProTyrTyrGlyArgGluGluGluLeuArgLysHis    165170175    ProIleLysArgAlaAlaGluGlyTrpAlaAlaProAspAlaLeuLeu    180185190    GlyGlnValLysAlaSerLeuLeuProGlyGlySerGluGlyGlyArg    195200205    ArgArgArgGluLeuAspProMetAspValArgGlySerIleValTyr    210215220    LeuGluIleAspAsnArgGlnCysValGlnAlaSerSerGlnCysPhe    225230235240    GlnSerAlaThrAspValAlaAlaPheLeuGlyAlaLeuAlaSerLeu    245250255    GlySerLeuAsnIleProTyrLysIleGluAlaValGlnSerGluThr    260265270    ValGluProProProProAlaGlnLeuHisPheMetTyrValAlaAla    275280285    AlaAlaPheValLeuLeuPhePheValGlyCysGlyValLeuLeuSer    290295300    ArgLysArgArgArgGlnHisGlyGlnLeuTrpPheProGluGlyPhe    305310315320    LysValSerGluAlaSerLysLysLysArgArgGluProLeuGlyGlu    325330335    AspSerValGlyLeuLysProLeuLysAsnAlaSerAspGlyAlaLeu    340345350    MetAspAspAsnGlnAsnGluTrpGlyAspGluAspLeuGluThrLys    355360365    LysPheArgPheGluGluProValValLeuProAspLeuAspAspGln    370375380    ThrAspHisArgGlnTrpThrGlnGlnHisLeuAspAlaAlaAspLeu    385390395400    ArgMetSerAlaMetAlaProThrProProGlnGlyGluValAspAla    405410415    AspCysMetAspValAsnValArgGlyProAspGlyPheThrProLeu    420425430    MetIleAlaSerCysSerGlyGlyGlyLeuGluThrGlyAsnSerGlu    435440445    GluGluGluAspAlaProAlaValIleSerAspPheIleTyrGlnGly    450455460    AlaSerLeuHisAsnGlnThrAspArgThrGlyGluThrAlaLeuHis    465470475480    LeuAlaAlaArgTyrSerArgSerAspAlaAlaLysArgLeuLeuGlu    485490495    AlaSerAlaAspAlaAsnIleGlnAspAsnMetGlyArgThrProLeu    500505510    HisAlaAlaValSerAlaAspAlaGlnGlyValPheGlnIleLeuIle    515520525    ArgAsnArgAlaThrAspLeuAspAlaArgMetHisAspGlyThrThr    530535540    ProLeuIleLeuAlaAlaArgLeuAlaValGluGlyMetLeuGluAsp    545550555560    LeuIleAsnSerHisAlaAspValAsnAlaValAspAspLeuGlyLys    565570575    SerAlaLeuHisTrpAlaAlaAlaValAsnAsnValAspAlaAlaVal    580585590    ValLeuLeuLysAsnGlyAlaAsnLysAspMetGlnAsnAsnArgGlu    595600605    GluThrProLeuPheLeuAlaAlaArgGluGlySerTyrGluThrAla    610615620    LysValLeuLeuAspHisPheAlaAsnArgAspIleThrAspHisMet    625630635640    AspArgLeuProArgAspIleAlaGlnGluArgMetHisHisAspIle    645650655    ValArgLeuLeuAspGluTyrAsnLeuValArgSerProGlnLeuHis    660665670    GlyAlaProLeuGlyGlyThrProThrLeuSerProProLeuCysSer    675680685    ProAsnGlyTyrLeuGlySerLeuLysProGlyValGlnGlyLysLys    690695700    ValArgLysProSerSerLysGlyLeuAlaCysGlySerLysGluAla    705710715720    LysAspLeuLysAlaArgArgLysLysSerGlnAspGlyLysGlyCys    725730735    LeuLeuAspSerSerGlyMetLeuSerProValAspSerLeuGluSer    740745750    ProHisGlyTyrLeuSerAspValAlaSerProProLeuLeuProSer    755760765    ProPheGlnGlnSerProSerValProLeuAsnHisLeuProGlyMet    770775780    ProAspThrHisLeuGlyIleGlyHisLeuAsnValAlaAlaLysPro    785790795800    GluMetAlaAlaLeuGlyGlyGlyGlyArgLeuAlaPheGluThrGly    805810815    ProProArgLeuSerHisLeuProValAlaSerGlyThrSerThrVal    820825830    LeuGlySerSerSerGlyGlyAlaLeuAsnPheThrValGlyGlySer    835840845    ThrSerLeuAsnGlyGlnCysGluTrpLeuSerArgLeuGlnSerGly    850855860    MetValProAsnGlnTyrAsnProLeuArgGlySerValAlaProGly    865870875880    ProLeuSerThrGlnAlaProSerLeuGlnHisGlyMetValGlyPro    885890895    LeuHisSerSerLeuAlaAlaSerAlaLeuSerGlnMetMetSerTyr    900905910    GlnGlyLeuProSerThrArgLeuAlaThrGlnProHisLeuValGln    915920925    ThrGlnGlnValGlnProGlnAsnLeuGlnMetGlnGlnGlnAsnLeu    930935940    GlnProAlaAsnIleGlnGlnGlnGlnSerLeuGlnProProProPro    945950955960    ProProGlnProHisLeuGlyValSerSerAlaAlaSerGlyHisLeu    965970975    GlyArgSerPheLeuSerGlyGluProSerGlnAlaAspValGlnPro    980985990    LeuGlyProSerSerLeuAlaValHisThrIleLeuProGlnGluSer    99510001005    ProAlaLeuProThrSerLeuProSerSerLeuValProProValThr    101010151020    AlaAlaGlnPheLeuThrProProSerGlnHisSerTyrSerSerPro    1025103010351040    ValAspAsnThrProSerHisGlnLeuGlnValProValProValMet    104510501055    ValMetIleArgSerSerAspProSerLysGlySerSerIleLeuIle    106010651070    GluAlaProAspSerTrp    1075    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4268 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 2..1972    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    GGAGGTGGATGTGTTAGATGTGAATGTCCGTGGCCCAGATGGCTGC46    GluValAspValLeuAspValAsnValArgGlyProAspGlyCys    151015    ACCCCATTGATGTTGGCTTCTCTCCGAGGAGGCAGCTCAGATTTGAGT94    ThrProLeuMetLeuAlaSerLeuArgGlyGlySerSerAspLeuSer    202530    GATGAAGATGAAGATGCAGAGGACTCTTCTGCTAACATCATCACAGAC142    AspGluAspGluAspAlaGluAspSerSerAlaAsnIleIleThrAsp    354045    TTGGTCTACCAGGGTGCCAGCCTCCAGGCCCAGACAGACCGGACTGGT190    LeuValTyrGlnGlyAlaSerLeuGlnAlaGlnThrAspArgThrGly    505560    GAGATGGCCCTGCACCTTGCAGCCCGCTACTCACGGGCTGATGCTGCC238    GluMetAlaLeuHisLeuAlaAlaArgTyrSerArgAlaAspAlaAla    657075    AAGCGTCTCCTGGATGCAGGTGCAGATGCCAATGCCCAGGACAACATG286    LysArgLeuLeuAspAlaGlyAlaAspAlaAsnAlaGlnAspAsnMet    80859095    GGCCGCTGTCCACTCCATGCTGCAGTGGCAGCTGATGCCCAAGGTGTC334    GlyArgCysProLeuHisAlaAlaValAlaAlaAspAlaGlnGlyVal    100105110    TTCCAGATTCTGATTCGCAACCGAGTAACTGATCTAGATGCCAGGATG382    PheGlnIleLeuIleArgAsnArgValThrAspLeuAspAlaArgMet    115120125    AATGATGGTACTACACCCCTGATCCTGGCTGCCCGCCTGGCTGTGGAG430    AsnAspGlyThrThrProLeuIleLeuAlaAlaArgLeuAlaValGlu    130135140    GGAATGGTGGCAGAACTGATCAACTGCCAAGCGGATGTGAATGCAGTG478    GlyMetValAlaGluLeuIleAsnCysGlnAlaAspValAsnAlaVal    145150155    GATGACCATGGAAAATCTGCTCTTCACTGGGCAGCTGCTGTCAATAAT526    AspAspHisGlyLysSerAlaLeuHisTrpAlaAlaAlaValAsnAsn    160165170175    GTGGAGGCAACTCTTTTGTTGTTGAAAAATGGGGCCAACCGAGACATG574    ValGluAlaThrLeuLeuLeuLeuLysAsnGlyAlaAsnArgAspMet    180185190    CAGGACAACAAGGAAGAGACACCTCTGTTTCTTGCTGCCCGGGAGGGG622    GlnAspAsnLysGluGluThrProLeuPheLeuAlaAlaArgGluGly    195200205    AGCTATGAAGCAGCCAAGATCCTGTTAGACCATTTTGCCAATCGAGAC670    SerTyrGluAlaAlaLysIleLeuLeuAspHisPheAlaAsnArgAsp    210215220    ATCACAGACCATATGGATCGTCTTCCCCGGGATGTGGCTCGGGATCGC718    IleThrAspHisMetAspArgLeuProArgAspValAlaArgAspArg    225230235    ATGCACCATGACATTGTGCGCCTTCTGGATGAATACAATGTGACCCCA766    MetHisHisAspIleValArgLeuLeuAspGluTyrAsnValThrPro    240245250255    AGCCCTCCAGGCACCGTGTTGACTTCTGCTCTCTCACCTGTCATCTGT814    SerProProGlyThrValLeuThrSerAlaLeuSerProValIleCys    260265270    GGGCCCAACAGATCTTTCCTCAGCCTGAAGCACACCCCAATGGGCAAG862    GlyProAsnArgSerPheLeuSerLeuLysHisThrProMetGlyLys    275280285    AAGTCTAGACGGCCCAGTGCCAAGAGTACCATGCCTACTAGCCTCCCT910    LysSerArgArgProSerAlaLysSerThrMetProThrSerLeuPro    290295300    AACCTTGCCAAGGAGGCAAAGGATGCCAAGGGTAGTAGGAGGAAGAAG958    AsnLeuAlaLysGluAlaLysAspAlaLysGlySerArgArgLysLys    305310315    TCTCTGAGTGAGAAGGTCCAACTGTCTGAGAGTTCAGTAACTTTATCC1006    SerLeuSerGluLysValGlnLeuSerGluSerSerValThrLeuSer    320325330335    CCTGTTGATTCCCTAGAATCTCCTCACACGTATGTTTCCGACACCACA1054    ProValAspSerLeuGluSerProHisThrTyrValSerAspThrThr    340345350    TCCTCTCCAATGATTACATCCCCTGGGATCTTACAGGCCTCACCCAAC1102    SerSerProMetIleThrSerProGlyIleLeuGlnAlaSerProAsn    355360365    CCTATGTTGGCCACTGCCGCCCCTCCTGCCCCAGTCCATGCCCAGCAT1150    ProMetLeuAlaThrAlaAlaProProAlaProValHisAlaGlnHis    370375380    GCACTATCTTTTTCTAACCTTCATGAAATGCAGCCTTTGGCACATGGG1198    AlaLeuSerPheSerAsnLeuHisGluMetGlnProLeuAlaHisGly    385390395    GCCAGCACTGTGCTTCCCTCAGTGAGCCAGTTGCTATCCCACCACCAC1246    AlaSerThrValLeuProSerValSerGlnLeuLeuSerHisHisHis    400405410415    ATTGTGTCTCCAGGCAGTGGCAGTGCTGGAAGCTTGAGTAGGCTCCAT1294    IleValSerProGlySerGlySerAlaGlySerLeuSerArgLeuHis    420425430    CCAGTCCCAGTCCCAGCAGATTGGATGAACCGCATGGAGGTGAATGAG1342    ProValProValProAlaAspTrpMetAsnArgMetGluValAsnGlu    435440445    ACCCAGTACAATGAGATGTTTGGTATGGTCCTGGCTCCAGCTGAGGGC1390    ThrGlnTyrAsnGluMetPheGlyMetValLeuAlaProAlaGluGly    450455460    ACCCATCCTGGCATAGCTCCCCAGAGCAGGCCACCTGAAGGGAAGCAC1438    ThrHisProGlyIleAlaProGlnSerArgProProGluGlyLysHis    465470475    ATAACCACCCCTCGGGAGCCCTTGCCCCCCATTGTGACTTTCCAGCTC1486    IleThrThrProArgGluProLeuProProIleValThrPheGlnLeu    480485490495    ATCCCTAAAGGCAGTATTGCCCAACCAGCGGGGGCTCCCCAGCCTCAG1534    IleProLysGlySerIleAlaGlnProAlaGlyAlaProGlnProGln    500505510    TCCACCTGCCCTCCAGCTGTTGCGGGCCCCCTGCCCACCATGTACCAG1582    SerThrCysProProAlaValAlaGlyProLeuProThrMetTyrGln    515520525    ATTCCAGAAATGGCCCGTTTGCCCAGTGTGGCTTTCCCCACTGCCATG1630    IleProGluMetAlaArgLeuProSerValAlaPheProThrAlaMet    530535540    ATGCCCCAGCAGGACGGGCAGGTAGCTCAGACCATTCTCCCAGCCTAT1678    MetProGlnGlnAspGlyGlnValAlaGlnThrIleLeuProAlaTyr    545550555    CATCCTTTCCCAGCCTCTGTGGGCAAGTACCCCACACCCCCTTCACAG1726    HisProPheProAlaSerValGlyLysTyrProThrProProSerGln    560565570575    CACAGTTATGCTTCCTCAAATGCTGCTGAGCGAACACCCAGTCACAGT1774    HisSerTyrAlaSerSerAsnAlaAlaGluArgThrProSerHisSer    580585590    GGTCACCTCCAGGGTGAGCATCCCTACCTGACACCATCCCCAGAGTCT1822    GlyHisLeuGlnGlyGluHisProTyrLeuThrProSerProGluSer    595600605    CCTGACCAGTGGTCAAGTTCATCACCCCACTCTGCTTCTGACTGGTCA1870    ProAspGlnTrpSerSerSerSerProHisSerAlaSerAspTrpSer    610615620    GATGTGACCACCAGCCCTACCCCTGGGGGTGCTGGAGGAGGTCAGCGG1918    AspValThrThrSerProThrProGlyGlyAlaGlyGlyGlyGlnArg    625630635    GGACCTGGGACACACATGTCTGAGCCACCACACAACAACATGCAGGTT1966    GlyProGlyThrHisMetSerGluProProHisAsnAsnMetGlnVal    640645650655    TATGCGTGAGAGAGTCCACCTCCAGTGTAGAGACATAACTGACTTTTGTAAATGCT2022    TyrAla    GCTGAGGAACAAATGAAGGTCATCCGGGAGAGAAATGAAGAAATCTCTGGAGCCAGCTTC2082    TAGAGGTAGGAAAGAGAAGATGTTCTTATTCAGATAATGCAAGAGAAGCAATTCGTCAGT2142    TTCACTGGGTATCTGCAAGGCTTATTGATTATTCTAATCTAATAAGACAAGTTTGTGGAA2202    ATGCAAGATGAATACAAGCCTTGGGTCCATGTTTACTCTCTTCTATTTGGAGAATAAGAT2262    GGATGCTTATTGAAGCCCAGACATTCTTGCAGCTTGGACTGCATTTTAAGCCCTGCAGGC2322    TTCTGCCATATCCATGAGAAGATTCTACACTAGCGTCCTGTTGGGAATTATGCCCTGGAA2382    TTCTGCCTGAATTGACCTACGCATCTCCTCCTCCTTGGACATTCTTTTGTCTTCATTTGG2442    TGCTTTTGGTTTTGCACCTCTCCGTGATTGTAGCCCTACCAGCATGTTATAGGGCAAGAC2502    CTTTGTGCTTTTGATCATTCTGGCCCATGAAAGCAACTTTGGTCTCCTTTCCCCTCCTGT2562    CTTCCCGGTATCCCTTGGAGTCTCACAAGGTTTACTTTGGTATGGTTCTCAGCACAAACC2622    TTTCAAGTATGTTGTTTCTTTGGAAAATGGACATACTGTATTGTGTTCTCCTGCATATAT2682    CATTCCTGGAGAGAGAAGGGGAGAAGAATACTTTTCTTCAACAAATTTTGGGGGCAGGAG2742    ATCCCTTCAAGAGGCTGCACCTTAATTTTTCTTGTCTGTGTGCAGGTCTTCATATAAACT2802    TTACCAGGAAGAAGGGTGTGAGTTTGTTGTTTTTCTGTGTATGGGCCTGGTCAGTGTAAA2862    GTTTTATCCTTGATAGTCTAGTTACTATGACCCTCCCCACTTTTTTAAAACCAGAAAAAG2922    GTTTGGAATGTTGGAATGACCAAGAGACAAGTTAACTCGTGCAAGAGCCAGTTACCCACC2982    CACAGGTCCCCCTACTTCCTGCCAAGCATTCCATTGACTGCCTGTATGGAACACATTTGT3042    CCCAGATCTGAGCATTCTAGGCCTGTTTCACTCACTCACCCAGCATATGAAACTAGTCTT3102    AACTGTTGAGCCTTTCCTTTCATATCCACAGAAGACACTGTCTCAAATGTTGTACCCTTG3162    CCATTTAGGACTGAACTTTCCTTAGCCCAAGGGACCCAGTGACAGTTGTCTTCCGTTTGT3222    CAGATGATCAGTCTCTACTGATTATCTTGCTGCTTAAAGGCCTGCTCACCAATCTTTCTT3282    TCACACCGTGTGGTCCGTGTTACTGGTATACCCAGTATGTTCTCACTGAAGACATGGACT3342    TTATATGTTCAAGTGCAGGAATTGGAAAGTTGGACTTGTTTTCTATGATCCAAAACAGCC3402    CTATAAGAAGGTTGGAAAAGGAGGAACTATATAGCAGCCTTTGCTATTTTCTGCTACCAT3462    TTCTTTTCCTCTGAAGCGGCCATGACATTCCCTTTGGCAACTAACGTAGAAACTCAACAG3522    AACATTTTCCTTTCCTAGAGTCACCTTTTAGATGATAATGGACAACTATAGACTTGCTCA3582    TTGTTCAGACTGATTGCCCCTCACCTGAATCCACTCTCTGTATTCATGCTCTTGGCAATT3642    TCTTTGACTTTCTTTTAAGGGCAGAAGCATTTTAGTTAATTGTAGATAAAGAATAGTTTT3702    CTTCCTCTTCTCCTTGGGCCAGTTAATAATTGGTCCATGGCTACACTGCAACTTCCGTCC3762    AGTGCTGTGATGCCCATGACACCTGCAAAATAAGTTCTGCCTGGGCATTTTGTAGATATT3822    AACAGGTGAATTCCCGACTCTTTTGGTTTGAATGACAGTTCTCATTCCTTCTATGGCTGC3882    AAGTATGCATCAGTGCTTCCCACTTACCTGATTTGTCTGTCGGTGGCCCCATATGGAAAC3942    CCTGCGTGTCTGTTGGCATAATAGTTTACAAATGGTTTTTTCAGTCCTATCCAAATTTAT4002    TGAACCAACAAAAATAATTACTTCTGCCCTGAGATAAGCAGATTAAGTTTGTTCATTCTC4062    TGCTTTATTCTCTCCATGTGGCAACATTCTGTCAGCCTCTTTCATAGTGTGCAAACATTT4122    TATCATTCTAAATGGTGACTCTCTGCCCTTGGACCCATTTATTATTCACAGATGGGGAGA4182    ACCTATCTGCATGGACCCTCACCATCCTCTGTGCAGCACACACAGTGCAGGGAGCCAGTG4242    GCGATGGCGATGACTTTCTTCCCCTG4268    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 657 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    GluValAspValLeuAspValAsnValArgGlyProAspGlyCysThr    151015    ProLeuMetLeuAlaSerLeuArgGlyGlySerSerAspLeuSerAsp    202530    GluAspGluAspAlaGluAspSerSerAlaAsnIleIleThrAspLeu    354045    ValTyrGlnGlyAlaSerLeuGlnAlaGlnThrAspArgThrGlyGlu    505560    MetAlaLeuHisLeuAlaAlaArgTyrSerArgAlaAspAlaAlaLys    65707580    ArgLeuLeuAspAlaGlyAlaAspAlaAsnAlaGlnAspAsnMetGly    859095    ArgCysProLeuHisAlaAlaValAlaAlaAspAlaGlnGlyValPhe    100105110    GlnIleLeuIleArgAsnArgValThrAspLeuAspAlaArgMetAsn    115120125    AspGlyThrThrProLeuIleLeuAlaAlaArgLeuAlaValGluGly    130135140    MetValAlaGluLeuIleAsnCysGlnAlaAspValAsnAlaValAsp    145150155160    AspHisGlyLysSerAlaLeuHisTrpAlaAlaAlaValAsnAsnVal    165170175    GluAlaThrLeuLeuLeuLeuLysAsnGlyAlaAsnArgAspMetGln    180185190    AspAsnLysGluGluThrProLeuPheLeuAlaAlaArgGluGlySer    195200205    TyrGluAlaAlaLysIleLeuLeuAspHisPheAlaAsnArgAspIle    210215220    ThrAspHisMetAspArgLeuProArgAspValAlaArgAspArgMet    225230235240    HisHisAspIleValArgLeuLeuAspGluTyrAsnValThrProSer    245250255    ProProGlyThrValLeuThrSerAlaLeuSerProValIleCysGly    260265270    ProAsnArgSerPheLeuSerLeuLysHisThrProMetGlyLysLys    275280285    SerArgArgProSerAlaLysSerThrMetProThrSerLeuProAsn    290295300    LeuAlaLysGluAlaLysAspAlaLysGlySerArgArgLysLysSer    305310315320    LeuSerGluLysValGlnLeuSerGluSerSerValThrLeuSerPro    325330335    ValAspSerLeuGluSerProHisThrTyrValSerAspThrThrSer    340345350    SerProMetIleThrSerProGlyIleLeuGlnAlaSerProAsnPro    355360365    MetLeuAlaThrAlaAlaProProAlaProValHisAlaGlnHisAla    370375380    LeuSerPheSerAsnLeuHisGluMetGlnProLeuAlaHisGlyAla    385390395400    SerThrValLeuProSerValSerGlnLeuLeuSerHisHisHisIle    405410415    ValSerProGlySerGlySerAlaGlySerLeuSerArgLeuHisPro    420425430    ValProValProAlaAspTrpMetAsnArgMetGluValAsnGluThr    435440445    GlnTyrAsnGluMetPheGlyMetValLeuAlaProAlaGluGlyThr    450455460    HisProGlyIleAlaProGlnSerArgProProGluGlyLysHisIle    465470475480    ThrThrProArgGluProLeuProProIleValThrPheGlnLeuIle    485490495    ProLysGlySerIleAlaGlnProAlaGlyAlaProGlnProGlnSer    500505510    ThrCysProProAlaValAlaGlyProLeuProThrMetTyrGlnIle    515520525    ProGluMetAlaArgLeuProSerValAlaPheProThrAlaMetMet    530535540    ProGlnGlnAspGlyGlnValAlaGlnThrIleLeuProAlaTyrHis    545550555560    ProPheProAlaSerValGlyLysTyrProThrProProSerGlnHis    565570575    SerTyrAlaSerSerAsnAlaAlaGluArgThrProSerHisSerGly    580585590    HisLeuGlnGlyGluHisProTyrLeuThrProSerProGluSerPro    595600605    AspGlnTrpSerSerSerSerProHisSerAlaSerAspTrpSerAsp    610615620    ValThrThrSerProThrProGlyGlyAlaGlyGlyGlyGlnArgGly    625630635640    ProGlyThrHisMetSerGluProProHisAsnAsnMetGlnValTyr    645650655    Ala    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 654 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    ThrProProGlnGlyGluIleGluAlaAspCysMetAspValAsnVal    151015    ArgGlyProAspGlyPheThrProLeuMetIleAlaSerCysSerGly    202530    GlyGlyLeuGluThrGlyAsnSerGluGluGluGluAspAlaSerAla    354045    AsnMetIleSerAspPheIleGlyGlnGlyAlaGlnLeuHisAsnGln    505560    ThrAspArgThrGlyGluThrAlaLeuHisLeuAlaAlaArgTyrAla    65707580    ArgAlaAspAlaAlaLysArgLeuLeuGluSerSerAlaAspAlaAsn    859095    ValGlnAspAsnMetGlyArgThrProLeuHisAlaAlaValAlaAla    100105110    AspAlaGlnGlyValPheGlnIleLeuIleArgAsnArgAlaThrAsp    115120125    LeuAspAlaArgMetPheAspGlyThrThrProLeuIleLeuAlaAla    130135140    ArgLeuAlaValGluGlyMetValGluGluLeuIleAsnAlaHisAla    145150155160    AspValAsnAlaValAspGluPheGlyLysSerAlaLeuHisTrpAla    165170175    AlaAlaValAsnAsnValAspAlaAlaAlaValLeuLeuLysAsnSer    180185190    AlaAsnLysAspMetGlnAsnAsnLysGluGluThrSerLeuPheLeu    195200205    AlaAlaArgGluGlySerTyrGluThrAlaLysValLeuLeuAspHis    210215220    TyrAlaAsnArgAspIleThrAspHisMetAspArgLeuProArgAsp    225230235240    IleAlaGlnGluArgMetHisHisAspIleValHisLeuLeuAspGlu    245250255    TyrAsnLeuValLysSerProThrLeuHisAsnGlyProLeuGlyAla    260265270    ThrThrLeuSerProProIleCysSerProAsnGlyTyrMetGlyAsn    275280285    MetLysProSerValGlnSerLysLysAlaArgLysProSerIleLys    290295300    GlyAsnGlyCysLysGluAlaLysGluLeuLysAlaArgArgLysLys    305310315320    SerGlnAspGlyLysThrThrLeuLeuAspSerGlySerSerGlyVal    325330335    LeuSerProValAspSerLeuGluSerThrHisGlyTyrLeuSerAsp    340345350    ValSerSerProProLeuMetThrSerProPheGlnGlnSerProSer    355360365    MetProLeuAsnHisLeuThrSerMetProGluSerGlnLeuGlyMet    370375380    AsnHisIleAsnMetAlaThrLysGlnGluMetAlaAlaGlySerAsn    385390395400    ArgMetAlaPheAspAlaMetValProArgLeuThrHisLeuAsnAla    405410415    SerSerProAsnThrIleMetSerAsnGlySerMetHisPheThrVal    420425430    GlyGlyAlaProThrMetAsnSerGlnCysAspTrpLeuAlaArgLeu    435440445    GlnAsnGlyMetValGlnAsnGlnTyrAspProIleArgAsnGlyIle    450455460    GlnGlnGlyAsnAlaGlnGlnAlaGlnAlaLeuGlnHisGlyLeuMet    465470475480    ThrSerLeuHisAsnGlyLeuProAlaThrThrLeuSerGlnMetMet    485490495    ThrTyrGlnAlaMetProAsnThrArgLeuAlaAsnGlnProHisLeu    500505510    MetGlnAlaGlnGlnMetGlnGlnGlnGlnAsnLeuGlnLeuHisGln    515520525    SerMetGlnGlnGlnHisHisAsnSerSerThrThrSerThrHisIle    530535540    AsnSerProPheCysSerSerAspIleSerGlnThrAspLeuGlnGln    545550555560    MetSerSerAsnAsnIleHisSerValMetProGlnAspThrGlnIle    565570575    PheAlaAlaSerLeuProSerAsnLeuThrGlnSerMetThrThrAla    580585590    GlnPheLeuThrProProSerGlnHisSerTyrSerSerProMetAsp    595600605    AsnThrProSerHisGlnLeuGlnValProAspHisProPheLeuThr    610615620    ProSerProGluSerProAspGlnTrpSerSerSerSerProHisSer    625630635640    AsnMetSerAspTrpSerGluGlyIleSerSerProProThr    645650    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 666 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    ThrProProGlnGlyGluValAspAlaAspCysMetAspValAsnVal    151015    ArgGlyProAspGlyPheThrProLeuMetIleAlaSerCysSerGly    202530    GlyGlyLeuGluThrGlyAsnSerGluGluGluGluAspAlaProAla    354045    ValIleSerAspPheIleTyrGlnGlyAlaSerLeuHisAsnGlnThr    505560    AspArgThrGlyGluThrAlaLeuHisLeuAlaAlaArgTyrSerArg    65707580    SerAspAlaAlaLysArgLeuLeuGluAlaSerAlaAspAlaAsnIle    859095    GlnAspAsnMetGlyArgThrProLeuHisAlaAlaValSerAlaAsp    100105110    AlaGlnGlyValPheGlnIleLeuLeuArgAsnArgAlaThrAspLeu    115120125    AspAlaArgMetHisAspGlyThrThrProLeuIleLeuAlaAlaArg    130135140    LeuAlaValGluGlyMetLeuGluAspLeuIleAsnSerHisAlaAsp    145150155160    ValAsnAlaValAspAspLeuGlyLysSerAlaLeuHisTrpAlaAla    165170175    AlaValAsnAsnValAspAlaAlaValValLeuLeuLysAsnGlyAla    180185190    AsnLysAspMetGlnAsnAsnLysGluGluThrProLeuPheLeuAla    195200205    AlaArgGluGlySerTyrGluThrAlaLysValLeuLeuAspHisPhe    210215220    AlaAsnArgAspIleThrAspHisMetAspArgLeuProArgAspIle    225230235240    AlaGlnGluArgMetHisHisAspIleValArgLeuLeuAspGluTyr    245250255    AsnLeuValArgSerProGlnLeuHisGlyThrAlaLeuGlyGlyThr    260265270    ProThrLeuSerProThrLeuCysSerProAsnGlyTyrLeuGlyAsn    275280285    LeuLysSerAlaThrGlnGlyLysLysAlaArgLysProSerThrLys    290295300    GlyLeuAlaCysSerSerLysGluAlaLysAspLeuLysAlaArgArg    305310315320    LysLysSerGlnAspGlyLysGlyCysLeuLeuAspSerSerSerMet    325330335    LeuSerProValAspSerLeuGluSerProHisGlyTyrLeuSerAsp    340345350    ValAlaSerProProLeuProSerProPheGlnGlnSerProSerMet    355360365    ProLeuSerHisLeuProGlyMetProAspThrHisLeuGlyIleSer    370375380    HisLeuAsnValAlaAlaLysProGluMetAlaAlaLeuAlaGlyGly    385390395400    SerArgLeuAlaPheGluProProProProArgLeuSerHisLeuPro    405410415    ValAlaSerSerAlaSerThrValLeuSerThrAsnGlyThrGlyAla    420425430    MetAsnPheThrValGlyAlaProAlaSerLeuAsnGlyGlnCysGlu    435440445    TrpLeuProArgLeuGlnAsnGlyMetValProSerGlnTyrAsnPro    450455460    LeuArgProGlyValThrProGlyThrLeuSerThrGlnAlaAlaGly    465470475480    LeuGlnHisGlyMetMetSerProIleHisSerSerLeuSerThrAsn    485490495    ThrLeuSerProIleIleTyrGlnGlyLeuProAsnThrArgLeuAla    500505510    ThrGlnProHisLeuValGlnThrGlnGlnValGlnProGlnAsnLeu    515520525    GlnIleGlnProGlnAsnLeuGlnProProSerGlnProHisLeuSer    530535540    ValSerSerAlaAlaAsnGlyHisLeuGlyArgSerPheLeuSerGly    545550555560    GluProSerGlnAlaAspValGlnProLeuGlyProSerSerLeuPro    565570575    ValHisThrIleLeuProGlnGluSerGlnAlaLeuProThrSerLeu    580585590    ProSerSerMetValProProMetThrThrThrGlnPheLeuThrPro    595600605    ProSerGlnHisSerTyrSerSerSerProValAspAsnThrProSer    610615620    HisGlnLeuGlnValProGluHisProPheLeuThrProSerProGlu    625630635640    SerProAspGlnTrpSerSerSerSerArgHisSerAsnIleSerAsp    645650655    TrpSerGluGlyIleSerSerProProThr    660665    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 681 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    ThrProProGlnGlyGluValAspAlaAspCysMetAspValAsnVal    151015    ArgGlyProAspGlyPheThrProLeuMetIleAlaSerCysSerGly    202530    GlyGlyLeuGluThrGlyAsnSerGluGluGluGluAspAlaProAla    354045    ValIleSerAspPheIleTyrGlnGlyAlaSerLeuHisAsnGlnThr    505560    AspArgThrGlyGluThrAlaLeuHisLeuAlaAlaArgTyrSerArg    65707580    SerAspAlaAlaLysArgLeuLeuGluAlaSerAlaAspAlaAsnIle    859095    GlnAspAsnMetGlyArgThrProLeuHisAlaAlaValSerAlaAsp    100105110    AlaGlnGlyValPheGlnIleLeuIleArgAsnArgAlaThrAspLeu    115120125    AspAlaArgMetHisAspGlyThrThrProLeuIleLeuAlaAlaArg    130135140    LeuAlaValGluGlyMetLeuGluAspLeuIleAsnSerHisAlaAsp    145150155160    ValAsnAlaValAspAspLeuGlyLysSerAlaLeuHisTrpAlaAla    165170175    AlaValAsnAsnValAspAlaAlaValValLeuLeuLysAsnGlyAla    180185190    AsnLysAspMetGlnAsnAsnArgGluGluThrProLeuPheLeuAla    195200205    AlaArgGluGlySerTyrGluThrAlaLysValLeuLeuAspHisPhe    210215220    AlaAsnArgAspIleThrAspHisMetAspArgLeuProArgAspIle    225230235240    AlaGlnGluArgMetHisHisAspIleValArgLeuLeuAspGluTyr    245250255    AsnLeuValArgSerProGlnLeuHisGlyAlaProLeuGlyGlyThr    260265270    ProThrLeuSerProProLeuCysSerProAsnGlyTyrLeuGlySer    275280285    LeuLysProGlyValGlnGlyLysLysValArgLysProSerSerLys    290295300    GlyLeuAlaCysGlySerLysGluAlaLysAspLeuLysAlaArgArg    305310315320    LysLysSerGlnAspGlyLysGlyCysLeuLeuAspSerSerGlyMet    325330335    LeuSerProValAspSerLeuGluSerProHisGlyTyrLeuSerAsp    340345350    ValAlaSerProProLeuLeuProSerProPheGlnGlnSerProSer    355360365    ValProLeuAsnHisLeuProGlyMetProAspThrHisLeuGlyIle    370375380    GlyHisLeuAsnValAlaAlaLysProGluMetAlaAlaLeuGlyGly    385390395400    GlyGlyArgLeuAlaPheGluThrGlyProProArgLeuSerHisLeu    405410415    ProValAlaSerGlyThrSerThrValLeuGlySerSerSerGlyGly    420425430    AlaLeuAsnPheThrValGlyGlySerThrSerLeuAsnGlyGlnCys    435440445    GluTrpLeuSerArgLeuGlnSerGlyMetValProAsnGlnTyrAsn    450455460    ProLeuArgGlySerValAlaProGlyProLeuSerThrGlnAlaPro    465470475480    SerLeuGlnHisGlyMetValGlyProLeuHisSerSerLeuAlaAla    485490495    SerAlaLeuSerGlnMetMetSerTyrGlnGlyLeuProSerThrArg    500505510    LeuAlaThrGlnProHisLeuValGlnThrGlnGlnValGlnProGln    515520525    AsnLeuGlnMetGlnGlnGlnAsnLeuGlnProAlaAsnIleGlnGln    530535540    GlnGlnSerLeuGlnProProProProProProGlnProHisLeuGly    545550555560    ValSerSerAlaAlaSerGlyHisLeuGlyArgSerPheLeuSerGly    565570575    GluProSerGlnAlaAspValGlnProLeuGlyProSerSerLeuAla    580585590    ValHisThrIleLeuProGlnGluSerProAlaLeuProThrSerLeu    595600605    ProSerSerLeuValProProValThrAlaAlaGlnPheLeuThrPro    610615620    ProSerGlnHisSerTyrSerSerProValGluAsnThrProSerHis    625630635640    GlnLeuGlnValProGluHisProPheLeuThrProSerProGluSer    645650655    ProAspGlnTrpSerSerSerSerProHisSerAsnValSerAspTrp    660665670    SerGluGlyValSerSerProProThr    675680    __________________________________________________________________________

What is claimed is:
 1. A substantially purified fragment of a Serrateprotein, which fragment is characterized by the ability in vitro, whenexpressed on the surface of a first cell, to bind to a Notch proteinexpressed on the surface of a second cell, which fragment lacks one ormore amino acids of the extracellular domain of the Serrate protein. 2.A substantially purified fragment of a Serrate protein which is theportion of the Serrate protein with the greatest homology over asequence of 199 amino acids to the amino acid sequence as depicted inFIGS. 15A-15B (SEQ ID NO:9) from about amino acid numbers 85-283, whichfragment lacks one or more amino acids of the extracellular domain ofthe Serrate protein.
 3. A chimeric protein comprising the fragment ofclaim 2 joined to a protein sequence of a second protein, in which saidsecond protein is not said Serrate protein, and in which the fragmentdoes not contain a complete EGF-homologous repeat of the Serrateprotein.
 4. A substantially purified fragment of a Serrate protein whichis the portion of the Serrate protein with the greatest homology over asequence of 204 amino acids, to the amino acid sequence as depicted inFIGS. 15A-15B (SEQ ID NO:9) from about amino acid numbers 79-282, whichfragment lacks one or more amino acids of the extracellular domain ofthe Serrate protein.
 5. The fragment of claim 1 which consists of atleast a portion of a Serrate protein with the greatest homology over asequence of 199 amino acids, to the amino acid depicted in FIGS. 15A-15B(SEQ ID NO:9) from about amino acid members 85-283.
 6. The fragment ofclaim 1, which consists of at least a portion of a Serrate protein withthe greatest homology over a sequence of 204 amino acids, to the aminoacid sequence as depicted in FIGS. 15A-15B (SEQ ID NO:9) from aboutamino acid numbers 79-282.
 7. A chimeric protein comprising the fragmentof claim 5 joined to a protein sequence of a second protein, in whichsaid second protein is not said Serrate protein, and in which thefragment does not contain a complete EFG-homologous repeat of theSerrate protein.
 8. A chimeric protein comprising the fragment of claim6 joined to a protein sequence of a second protein, in which said secondprotein is not said Serrate protein, and in which the fragment does notcontain a complete EFG-homologous repeat of the Serrate protein.
 9. Thefragment of claim 1 which consists of at least the amino acid sequenceas depicted in FIGS. 15A-15B (SEQ ID NO:9) from about amino acid numbers85-283.
 10. The fragment of claim 1 which consists of at least the aminoacid sequence as depicted in FIGS. 15A-15B (SEQ ID NO:9) from aboutamino acid numbers 79-282.
 11. A derivative of the fragment of claim 1,which derivative is characterized by the ability in vitro, whenexpressed on the surface of a first cell, to bind to a Notch proteinexpressed on the surface of a second cell, the derivative having aninsertion, substitution or deletion of one or more amino acids relativeto the fragment, said derivative lacking one or more amino acids of theextracellular domain of the Serrate protein.
 12. The fragment of claims1, 2, 4, 5, 6, 9 or 10, which does not contain a complete EGF-homologousrepeat of the Serrate protein.
 13. The derivative of claim 11 which doesnot contain a complete EGF-homologous repeat of the Serrate protien. 14.A substantially pure derivative of a Serrate protein, which derivativeis characterized by the ability in vitro, when expressed on the surfaceof a first cell, to bind to a Notch protein expressed on the surface ofa second cell, the derivative having an insertion, substitution, ordeletion of one or more amino acids in at least the extracellular domainof the Serrate protein.
 15. The derivative of claim 14 in which theinsertion, substitution, or deletion is in at least a portion of anEFG-homologous repeat.
 16. A chimeric protein comprising the derivativeof claims 11, 13, 14 or 15 joined to a protein sequence of a secondprotein, in which said second protein is not said Serrate protein.
 17. Achimeric protein comprising the fragment of claim 1 joined to a proteinsequence of a second protien, in which said second protein is not saidSerrate protein.
 18. A substantially purified fragment of a Serrateprotein consisting essentially of amino acid numbers 79-282 of the aminoacid sequence as depicted in FIGS. 15A-15B (SEQ ID NO:9), which fragmentis characterized by the ability in vitro, when expressed on the surfaceof a first cell, to bind to a Notch protein expressed on the surface ofa second cell.
 19. A substantially purified fragment of a Serrateprotein consisting essentially of amino acid numbers 85-283 of the aminoacid sequence as depicted in FIGS. 15A-15B (SEQ ID NO:9), which fragmentis characterized by the ability in vitro, when expressed on the surfaceof a first cell, to bind to a Notch protein expressed on the surface ofa second cell.